Cloning of cytochrome p450 genes from nicotiana

ABSTRACT

The present invention relates to p450 enzymes and nucleic acid sequences encoding p450 enzymes in  Nicotiana , and methods of using those enzymes and nucleic acid sequences to alter plant phenotypes.

The present invention relates to nucleic acid sequences encodingcytochrome p450 enzymes (hereinafter referred to as p450 and p450enzymes) in Nicotiana plants and methods for using those nucleic acidsequences to alter plant phenotypes.

BACKGROUND

Cytochrome p450s catalyze enzymatic reactions for a diverse range ofchemically dissimilar substrates that include the oxidative,peroxidative and reductive metabolism of endogenous and xenobioticsubstrates. In plants, p450s participate in biochemical pathways thatinclude the synthesis of plant products such as phenylpropanoids,alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates(Chappel, Annu. Rev. Plant Physiol. Plant Mol. Biol. 198, 49:311-343).Cytochrome p450s, also known as p450 heme-thiolate proteins, usually actas terminal oxidases in multi-component electron transfer chains, calledp450-containing monooxygenase systems. Specific reactions catalyzedinclude demethylation, hydroxylation, epoxidation, N-oxidation,sulfooxidation, N-, S-, and O-dealkylations, desulfation, deamination,and reduction of azo, nitro, and N-oxide groups.

The diverse role of Nicotiana plant p450 enzymes has been implicated ineffecting a variety of plant metabolites such as phenylpropanoids,alkaloids, terpenoids, lipids, cyanogenic glycosides, glucosinolates anda host of other chemical entities. During recent years, it is becomingapparent that some p450 enzymes can impact the composition of plantmetabolites in plants. For example, it has been long desired to improvethe flavor and aroma of certain plants by altering its profile ofselected fatty acids through breeding; however very little is knownabout mechanisms involved in controlling the levels of these leafconstituents. The down regulation of p450 enzymes associated with themodification of fatty acids may facilitate accumulation of desired fattyacids that provide more preferred leaf phenotypic qualities. Thefunction of p450 enzymes and their broadening roles in plantconstituents is still being discovered. For instance, a special class ofp450 enzymes was found to catalyze the breakdown of fatty acid intovolatile C6- and C9-aldehydes and -alcohols that are major contributorsof “fresh green” odor of fruits and vegetables. The level of other noveltargeted p450s may be altered to enhance the qualities of leafconstituents by modifying lipid composition and related break downmetabolites in Nicotiana leaf. Several of these constituents in leaf areaffected by senescence that stimulates the maturation of leaf qualityproperties. Still other reports have shown that p450s enzymes are play afunctional role in altering fatty acids that are involved inplant-pathogen interactions and disease resistance.

In other instances, p450 enzymes have been suggested to be involved inalkaloid biosynthesis. Nornicotine is a minor alkaloid found inNicotiana tabaceum. It has been postulated that it is produced by thep450 mediated demethylation of nicotine followed by acylation andnitrosation at the N position thereby producing a series ofN-acylnonicotines and N-nitrosonornicotines. N-demethylation, catalyzedby a putative p450 demethylase, is thought to be a primary source ofnornicotine biosyntheses in Nicotiana. While the enzyme is believed tobe microsomal, thus far a nicotine demethylase enzyme has not beensuccessfully purified, nor have the genes involved been isolated.

Furthermore, it is hypothesized but not proven that the activity of p450enzymes is genetically controlled and also strongly influenced byenvironment factors. For example, the demethylation of nicotine inNicotiana is thought to increase substantially when the plants reach amature stage. Furthermore, it is hypothesized yet not proven that thedemethylase gene contains a transposable element that can inhibittranslation of RNA when present.

The large multiplicity of p450 enzyme forms, their differing structureand function have made their research on Nicotiana p450 enzymes verydifficult before the enclosed invention. In addition, cloning of p450enzymes has been hampered at least in part because thesemembrane-localized proteins are typically present in low abundance andoften unstable to purification. Hence, a need exists for theidentification of p450 enzymes in plants and the nucleic acid sequencesassociated with those p450 enzymes. In particular, only a few cytochromep450 proteins have been reported in Nicotiana. The inventions describedherein entail the discovery of a substantial number of cytochrome p450fragments that correspond to several groups of p450 species based ontheir sequence identity.

SUMMARY

The present invention is directed to plant p450 enzymes. The presentinvention is further directed to plant p450 enzymes from Nicotiana. Thepresent invention is also directed to p450 enzymes in plants whoseexpression is induced by ethylene and/or plant senescence. The presentinvention is yet further directed to nucleic acid sequences in plantshaving enzymatic activities, for example, being categorized asoxygenase, demethylase and the like, or other and the use of thosesequences to reduce or silence the expression or over-expression ofthese enzymes. The invention also relates to p450 enzymes found inplants containing higher nornicotine levels than plants exhibiting lowernornicotine levels.

In one aspect, the invention is directed to nucleic acid sequences asset forth in SEQ. ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295 and297.

In a second related aspect, those fragments containing greater than 75%identity in nucleic acid sequence were placed into groups dependent upontheir identity in a region corresponding to the first nucleic acidfollowing the cytochrome p450 motif GXRXCX(G/A) to the stop codon. Therepresentative nucleic acid groups and respective species are shown inTable I.

In a third aspect, the invention is directed to amino acid sequences asset forth in SEQ. ID. Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296 and298.

In a fourth related aspect, those fragments containing greater than 71%identity in amino acid sequence were placed into groups dependent upontheir identity to each other in a region corresponding to the firstamino acid following the cytochrome p450 motif GXRXCX(G/A) to the stopcodon. The representative amino acid groups and respective species areshown in Table II.

In a fifth aspect, the invention is directed to amino acid sequences offull length genes as set forth in SEQ. ID. Nos. 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296 and 298.

In a sixth related aspect, those full length genes containing 85% orgreater identity in amino acid sequence were placed into groupsdependent upon the identity to each other. The representative amino acidgroups and respective species are shown in Table III.

In a seventh aspect, the invention is directed to amino acid sequencesof the fragments set forth in SEQ. ID. Nos. 299-357.

In the eighth related aspect, those fragments containing 90% or greateridentity in amino acid sequence were placed into groups dependent upontheir identity to each other in a region corresponding to the firstcytochrome p450 domain, UXXRXXZ, to the third cytochrome domain, GXRXO,where U is E or K, X is any amino acid and Z is R, T, S or M. Therepresentative amino acid groups respective species shown in Table IV.

In a ninth related aspect, the reduction or elimination orover-expression of p450 enzymes in Nicotiana plants may be accomplishedtransiently using RNA viral systems.

Resulting transformed or infected plants are assessed for phenotypicchanges including, but not limited to, analysis of endogenous p450 RNAtranscripts, p450 expressed peptides, and concentrations of plantmetabolites using techniques commonly available to one having ordinaryskill in the art.

In a tenth important aspect, the present invention is also directed togeneration of trangenic Nicotiana lines that have altered p450 enzymeactivity levels. In accordance with the invention, these transgeniclines include nucleic acid sequences that are effective for reducing orsilencing or increasing the expression of certain enzyme thus resultingin phenotypic effects within Nicotiana. Such nucleic acid sequencesinclude SEQ. ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295 and 297.

In a very important eleventh aspect of the invention, plant cultivarsincluding nucleic acids of the present invention in a down regulationcapacity using either full length genes or fragments thereof or in anover-expression capacity using full length genes will have alteredmetabolite profiles relative to control plants.

In a twelfth aspect of the invention, plant cultivars including nucleicacid of the present invention using either full length genes orfragments thereof in modifying the biosynthesis or breakdown ofmetabolites derived from the plant or external to the plants, will haveuse in tolerating certain exogenous chemicals or plant pests. Suchnucleic acid sequences include SEQ ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289,291, 293, 295 and 297.

In a thirteenth aspect, the present invention is directed to thescreening of plants, more preferably Nicotiana, that contain genes thathave substantial nucleic acid identity to the taught nucleic acidsequence. The use of the invention would be advantageous to identify andselect plants that contain a nucleic acid sequence with exact orsubstantial identity where such plants are part of a breeding programfor traditional or transgenic varieties, a mutagenesis program, ornaturally occurring diverse plant populations. The screening of plantsfor substantial nucleic acid identity may be accomplished by evaluatingplant nucleic acid materials using a nucleic acid probe in conjunctionwith nucleic acid detection protocols including, but not limited to,nucleic acid hybridization and PCR analysis. The nucleic acid probe mayconsist of the taught nucleic acid sequence or fragment thereofcorresponding to SEQ ID 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241,243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269,271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295 and 297.

In a fourteenth aspect, the present invention is directed to theidentification of plant genes, more preferably Nicotiana, that sharesubstantial amino acid identity corresponding to the taught nucleic acidsequence. The identification of plant genes including both cDNA andgenomic clones, those cDNAs and genomic clones, more preferably fromNicotiana may be accomplished by screening plant cDNA libraries using anucleic acid probe in conjunction with nucleic acid detection protocolsincluding, but not limited to, nucleic acid hybridization and PCRanalysis. The nucleic acid probe may be comprised of nucleic acidsequence or fragment thereof corresponding to SEQ ID 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 143, 145 and 147.

In an alterative fifteenth aspect, cDNA expression libraries thatexpress peptides may be screened using antibodies directed to part orall of the taught amino acid sequence. Such amino acid sequences includeSEQ ID 2, 4, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 144, 146, 148.

In a sixteenth important aspect, the present invention is also directedto generation of transgenic Nicotiana lines that have over-expression ofp450 enzyme activity levels. In accordance with the invention, thesetransgenic lines include all nucleic acid sequences encoding the aminoacid sequences of full length genes that are effective for increasingthe expression of certain enzyme thus resulting in phenotypic effectswithin Nicotiana. Such amino acid sequences include SEQ. ID. 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296 and 298.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows nucleic acid SEQ. ID. No.:1 and amino acid SEQ. ID. No.:2.

FIG. 2 shows nucleic acid SEQ. ID. No.:3 and amino acid SEQ. ID. No.:4.

FIG. 3 shows nucleic acid SEQ. ID. No.:5 and amino acid SEQ. ID. No.:6.

FIG. 4 shows nucleic acid SEQ. ID. No.:7 and amino acid SEQ. ID. No.:8.

FIG. 5 shows nucleic acid SEQ. ID. No.:9 and amino acid SEQ. ID. No.:10.

FIG. 6 shows nucleic acid SEQ. ID. No.:11 and amino acid SEQ. ID.No.:12.

FIG. 7 shows nucleic acid SEQ. ID. No.:13 and amino acid SEQ. ID.No.:14.

FIG. 8 shows nucleic acid SEQ. ID. No.:15 and amino acid SEQ. ID.No.:16.

FIG. 9 shows nucleic acid SEQ. ID. No.:17 and amino acid SEQ. ID.No.:18.

FIG. 10 shows nucleic acid SEQ. ID. No.:19 and amino acid SEQ. ID.No.:20.

FIG. 11 shows nucleic acid SEQ. ID. No.:21 and amino acid SEQ. ID.No.:22.

FIG. 12 shows nucleic acid SEQ. ID. No.:23 and amino acid SEQ. ID.No.:24.

FIG. 13 shows nucleic acid SEQ. ID. No.:25 and amino acid SEQ. ID.No.:26.

FIG. 14 shows nucleic acid SEQ. ID. No.:27 and amino acid SEQ. ID.No.:28.

FIG. 15 shows nucleic acid SEQ. ID. No.:29 and amino acid SEQ. ID.No.:30.

FIG. 16 shows nucleic acid SEQ. ID. No.:31 and amino acid SEQ. ID.No.:32.

FIG. 17 shows nucleic acid SEQ. ID. No.:33 and amino acid SEQ. ID.No.:34.

FIG. 18 shows nucleic acid SEQ. ID. No.:35 and amino acid SEQ. ID.No.:36.

FIG. 19 shows nucleic acid SEQ. ID. No.:37 and amino acid SEQ. ID.No.:38.

FIG. 20 shows nucleic acid SEQ. ID. No.:39 and amino acid SEQ. ID.No.:40.

FIG. 21 shows nucleic acid SEQ. ID. No.:41 and amino acid SEQ. ID.No.:42.

FIG. 22 shows nucleic acid SEQ. ID. No.:43 and amino acid SEQ. ID.No.:44.

FIG. 23 shows nucleic acid SEQ. ID. No.:45 and amino acid SEQ. ID.No.:46.

FIG. 24 shows nucleic acid SEQ. ID. No.:47 and amino acid SEQ. ID.No.:48.

FIG. 25 shows nucleic acid SEQ. ID. No.:49 and amino acid SEQ. ID.No.:50.

FIG. 26 shows nucleic acid SEQ. ID. No.:51 and amino acid SEQ. ID.No.:52.

FIG. 27 shows nucleic acid SEQ. ID. No.:53 and amino acid SEQ. ID.No.:54.

FIG. 28 shows nucleic acid SEQ. ID. No.:55 and amino acid SEQ. ID.No.:56.

FIG. 29 shows nucleic acid SEQ. ID. No.:57 and amino acid SEQ. ID.No.:58.

FIG. 30 shows nucleic acid SEQ. ID. No.:59 and amino acid SEQ. ID.No.:60.

FIG. 31 shows nucleic acid SEQ. ID. No.:61 and amino acid SEQ. ID.No.:62.

FIG. 32 shows nucleic acid SEQ. ID. No.:63 and amino acid SEQ. ID.No.:64.

FIG. 33 shows nucleic acid SEQ. ID. No.:65 and amino acid SEQ. ID.No.:66.

FIG. 34 shows nucleic acid SEQ. ID. No.:67 and amino acid SEQ. ID.No.:68.

FIG. 35 shows nucleic acid SEQ. ID. No.:69 and amino acid SEQ. ID.No.:70.

FIG. 36 shows nucleic acid SEQ. ID. No.:71 and amino acid SEQ. ID.No.:72.

FIG. 37 shows nucleic acid SEQ. ID. No.:73 and amino acid SEQ. ID.No.:74.

FIG. 38 shows nucleic acid SEQ. ID. No.:75 and amino acid SEQ. ID.No.:76.

FIG. 39 shows nucleic acid SEQ. ID. No.:77 and amino acid SEQ. ID.No.:78.

FIG. 40 shows nucleic acid SEQ. ID. No.:79 and amino acid SEQ. ID.No.:80.

FIG. 41 shows nucleic acid SEQ. ID. No.:81 and amino acid SEQ. ID.No.:82.

FIG. 42 shows nucleic acid SEQ. ID. No.:83 and amino acid SEQ. ID.No.:84.

FIG. 43 shows nucleic acid SEQ. ID. No.:85 and amino acid SEQ. ID.No.:86.

FIG. 44 shows nucleic acid SEQ. ID. No.:87 and amino acid SEQ. ID.No.:88.

FIG. 45 shows nucleic acid SEQ. ID. No.:89 and amino acid SEQ. ID.No.:90.

FIG. 46 shows nucleic acid SEQ. ID. No.:91 and amino acid SEQ. ID.No.:92.

FIG. 48 shows nucleic acid SEQ. ID. No.:95 and amino acid SEQ. ID.No.:96.

FIG. 49 shows nucleic acid SEQ. ID. No.:97 and amino acid SEQ. ID.No.:98.

FIG. 50 shows nucleic acid SEQ. ID. No.:99 and amino acid SEQ. ID.No.:100.

FIG. 51 shows nucleic acid SEQ. ID. No.:101 and amino acid SEQ. ID.No.:102.

FIG. 52 shows nucleic acid SEQ. ID. No.:103 and amino acid SEQ. ID.No.:104.

FIG. 53 shows nucleic acid SEQ. ID. No.:105 and amino acid SEQ. ID.No.:106.

FIG. 54 shows nucleic acid SEQ. ID. No.:107 and amino acid SEQ. ID.No.:108.

FIG. 55 shows nucleic acid SEQ. ID. No.:109 and amino acid SEQ. ID.No.:110.

FIG. 56 shows nucleic acid SEQ. ID. No.:111 and amino acid SEQ. ID.No.:112.

FIG. 57 shows nucleic acid SEQ. ID. No.:113 and amino acid SEQ. ID.No.:114.

FIG. 58 shows nucleic acid SEQ. ID. No.:115 and amino acid SEQ. ID.No.:116.

FIG. 59 shows nucleic acid SEQ. ID. No.:117 and amino acid SEQ. ID.No.:118.

FIG. 60 shows nucleic acid SEQ. ID. No.:119 and amino acid SEQ. ID.No.:120.

FIG. 61 shows nucleic acid SEQ. ID. No.:121 and amino acid SEQ. ID.No.:122.

FIG. 62 shows nucleic acid SEQ. ID. No.:123 and amino acid SEQ. ID.No.:124.

FIG. 63 shows nucleic acid SEQ. ID. No.:125 and amino acid SEQ. ID.No.:126.

FIG. 64 shows nucleic acid SEQ. ID. No.:127 and amino acid SEQ. ID.No.:128.

FIG. 65 shows nucleic acid SEQ. ID. No.:129 and amino acid SEQ. ID.No.:130.

FIG. 66 shows nucleic acid SEQ. ID. No.:131 and amino acid SEQ. ID.No.:132.

FIG. 67 shows nucleic acid SEQ. ID. No.:133 and amino acid SEQ. ID.No.:134.

FIG. 68 shows nucleic acid SEQ. ID. No.:135 and amino acid SEQ. ID.No.:136.

FIG. 69 shows nucleic acid SEQ. ID. No.:137 and amino acid SEQ. ID.No.:138.

FIG. 70 shows nucleic acid SEQ. ID. No.:139 and amino acid SEQ. ID.No.:140.

FIG. 72 shows nucleic acid SEQ. ID. No.:143 and amino acid SEQ. ID.No.:144.

FIG. 73 shows nucleic acid SEQ. ID. No.:145 and amino acid SEQ. ID.No.:146.

FIG. 74 shows nucleic acid SEQ. ID. No.:147 and amino acid SEQ. ID.No.:148.

FIG. 75 shows nucleic acid SEQ. ID No.: 149 and amino acid SEQ. ID. No.:150.

FIG. 76 shows nucleic acid SEQ. ID No.: 151 and amino acid SEQ. ID. No.:152.

FIG. 77 shows nucleic acid SEQ. ID No.: 153 and amino acid SEQ. ID. No.:154.

FIG. 78 shows nucleic acid SEQ. ID No.: 155 and amino acid SEQ. ID. No.:156.

FIG. 79 shows nucleic acid SEQ. ID No.: 157 and amino acid SEQ. ID. No.:158.

FIG. 80 shows nucleic acid SEQ. ID No.: 159 and amino acid SEQ. ID. No.:160.

FIG. 81 shows nucleic acid SEQ. ID No.: 161 and amino acid SEQ. ID. No.:162.

FIG. 82 shows nucleic acid SEQ. ID No.: 163 and amino acid SEQ. ID. No.:164.

FIG. 83 shows nucleic acid SEQ. ID No.: 165 and amino acid SEQ. ID. No.:166.

FIG. 84 shows nucleic acid SEQ. ID No.: 167 and amino acid SEQ. ID. No.:168.

FIG. 85 shows nucleic acid SEQ. ID No.: 169 and amino acid SEQ. ID. No.:170.

FIG. 86 shows nucleic acid SEQ. ID No.: 171 and amino acid SEQ. ID. No.:172.

FIG. 87 shows nucleic acid SEQ. ID No.: 173 and amino acid SEQ. ID. No.:174.

FIG. 88 shows nucleic acid SEQ. ID No.: 175 and amino acid SEQ. ID. No.:176.

FIG. 89 shows nucleic acid SEQ. ID No.: 177 and amino acid SEQ. ID. No.:178.

FIG. 90 shows nucleic acid SEQ. ID No.: 179 and amino acid SEQ. ID. No.:180.

FIG. 91 shows nucleic acid SEQ. ID No.: 181 and amino acid SEQ. ID. No.:182.

FIG. 92 shows nucleic acid SEQ. ID No.: 183 and amino acid SEQ. ID. No.:184.

FIG. 93 shows nucleic acid SEQ. ID No.: 185 and amino acid SEQ. ID. No.:186.

FIG. 94 shows nucleic acid SEQ. ID No.: 187 and amino acid SEQ. ID. No.:188.

FIG. 95 shows nucleic acid SEQ. ID No.: 189 and amino acid SEQ. ID. No.:190.

FIG. 96 shows nucleic acid SEQ. ID No.: 191 and amino acid SEQ. ID. No.:192.

FIG. 97 shows nucleic acid SEQ. ID No.: 193 and amino acid SEQ. ID. No.:194.

FIG. 98 shows nucleic acid SEQ. ID No.: 195 and amino acid SEQ. ID. No.:196.

FIG. 99 shows nucleic acid SEQ. ID No.: 197 and amino acid SEQ. ID. No.:198.

FIG. 100 shows nucleic acid SEQ. ID No.: 199 and amino acid SEQ. ID.No.: 200.

FIG. 101 shows nucleic acid SEQ. ID No.: 201 and amino acid SEQ. ID.No.: 202.

FIG. 102 shows nucleic acid SEQ. ID No.: 203 and amino acid SEQ. ID.No.: 204.

FIG. 103 shows nucleic acid SEQ. ID No.: 205 and amino acid SEQ. ID.No.: 206.

FIG. 104 shows nucleic acid SEQ. ID No.: 207 and amino acid SEQ. ID.No.: 208.

FIG. 105 shows nucleic acid SEQ. ID No.: 209 and amino acid SEQ. ID.No.: 210.

FIG. 106 shows nucleic acid SEQ. ID No.: 211 and amino acid SEQ. ID.No.: 212.

FIG. 107 shows nucleic acid SEQ. ID No.: 213 and amino acid SEQ. ID.No.: 214.

FIG. 108 shows nucleic acid SEQ. ID No.: 215 and amino acid SEQ. ID.No.: 216.

FIG. 109 shows nucleic acid SEQ. ID No.: 217 and amino acid SEQ. ID.No.: 218.

FIG. 110 shows nucleic acid SEQ. ID No.: 219 and amino acid SEQ. ID.No.: 220.

FIG. 111 shows nucleic acid SEQ. ID No.: 221 and amino acid SEQ. ID.No.: 222.

FIG. 112 shows nucleic acid SEQ. ID No.: 223 and amino acid SEQ. ID.No.: 224.

FIG. 113 shows nucleic acid SEQ. ID No.: 225 and amino acid SEQ. ID.No.: 226.

FIG. 114 shows nucleic acid SEQ. ID No.: 227 and amino acid SEQ. ID.No.: 228.

FIG. 115 shows nucleic acid SEQ. ID No.: 229 and amino acid SEQ. ID.No.: 230.

FIG. 116 shows nucleic acid SEQ. ID No.: 231 and amino acid SEQ. ID.No.: 232.

FIG. 117 shows nucleic acid SEQ. ID No.: 233 and amino acid SEQ. ID.No.: 234.

FIG. 118 shows nucleic acid SEQ. ID No.: 235 and amino acid SEQ. ID.No.: 236.

FIG. 119 shows nucleic acid SEQ. ID No.: 237 and amino acid SEQ. ID.No.: 238.

FIG. 120 shows nucleic acid SEQ. ID No.: 239 and amino acid SEQ. ID.No.: 240.

FIG. 121 shows nucleic acid SEQ. ID No.: 241 and amino acid SEQ. ID.No.: 242.

FIG. 122 shows nucleic acid SEQ. ID No.: 243 and amino acid SEQ. ID.No.: 244.

FIG. 123 shows nucleic acid SEQ. ID No.: 245 and amino acid SEQ. ID.No.: 246.

FIG. 124 shows nucleic acid SEQ. ID No.: 247 and amino acid SEQ. ID.No.: 248.

FIG. 125 shows nucleic acid SEQ. ID No.: 249 and amino acid SEQ. ID.No.: 250.

FIG. 126 shows nucleic acid SEQ. ID No.: 251 and amino acid SEQ. ID.No.: 252.

FIG. 127 shows nucleic acid SEQ. ID No.: 253 and amino acid SEQ. ID.No.: 254.

FIG. 128 shows nucleic acid SEQ. ID No.: 255 and amino acid SEQ. ID.No.: 256.

FIG. 129 shows nucleic acid SEQ. ID No.: 257 and amino acid SEQ. ID.No.: 258.

FIG. 130 shows nucleic acid SEQ. ID No.: 259 and amino acid SEQ. ID.No.: 260.

FIG. 131 shows nucleic acid SEQ. ID No.: 261 and amino acid SEQ. ID.No.: 262.

FIG. 132 shows nucleic acid SEQ. ID No.: 263 and amino acid SEQ. ID.No.: 264.

FIG. 133 shows nucleic acid SEQ. ID No.: 265 and amino acid SEQ. ID.No.: 266.

FIG. 134 shows nucleic acid SEQ. ID No.: 267 and amino acid SEQ. ID.No.: 268.

FIG. 135 shows nucleic acid SEQ. ID No.: 269 and amino acid SEQ. ID.No.: 270.

FIG. 136 shows nucleic acid SEQ. ID No.: 271 and amino acid SEQ. ID.No.: 272.

FIG. 137 shows nucleic acid SEQ. ID No.: 273 and amino acid SEQ. ID.No.: 274.

FIG. 138 shows nucleic acid SEQ. ID No.: 275 and amino acid SEQ. ID.No.: 276.

FIG. 139 shows nucleic acid SEQ. ID No.: 277 and amino acid SEQ. ID.No.: 278.

FIG. 140 shows nucleic acid SEQ. ID No.: 279 and amino acid SEQ. ID.No.: 280.

FIG. 141 shows nucleic acid SEQ. ID No.: 281 and amino acid SEQ. ID.No.: 282.

FIG. 142 shows nucleic acid SEQ. ID No.: 283 and amino acid SEQ. ID.No.: 284.

FIG. 143 shows nucleic acid SEQ. ID No.: 285 and amino acid SEQ. ID.No.: 286.

FIG. 144 shows nucleic acid SEQ. ID No.: 287 and amino acid SEQ. ID.No.: 288.

FIG. 145 shows nucleic acid SEQ. ID No.: 289 and amino acid SEQ. ID.No.: 290.

FIG. 146 shows nucleic acid SEQ. ID No.: 291 and amino acid SEQ. ID.No.: 292.

FIG. 147 shows nucleic acid SEQ. ID No.: 293 and amino acid SEQ. ID.No.: 294.

FIG. 148 shows nucleic acid SEQ. ID No.: 295 and amino acid SEQ. ID.No.: 296.

FIG. 149 shows nucleic acid SEQ. ID No.: 297 and amino acid SEQ. ID.No.: 298.

FIG. 151 shows a comparison of Sequence Groups.

FIG. 152 illustrates alignment of full length clones.

FIG. 153 shows a procedure used for cloning of cytochrome p450 cDNAfragments by PCR

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al. (1994)Dictionary of Microbiology and Molecular Biology, second edition, JohnWiley and Sons (New York) provides one of skill with a generaldictionary of many of the terms used in this invention. All patents andpublications referred to herein are incorporated by reference herein.For purposes of the present invention, the following terms are definedbelow.

“Enzymatic activity” is meant to include demethylation, hydroxylation,epoxidation, N-oxidation, sulfooxidation, N-, S-, and O-dealkylations,desulfation, deamination, and reduction of azo, nitro, and N-oxidegroups. The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, orsense or anti-sense, and unless otherwise limited, encompasses knownanalogues of natural nucleotides that hybridize to nucleic acids in amanner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence includes the complementarysequence thereof.

The terms “operably linked”, “in operable combination”, and “in operableorder” refer to functional linkage between a nucleic acid expressioncontrol sequence (such as a promoter, signal sequence, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence affects transcription and/ortranslation of the nucleic acid corresponding to the second sequence.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, expresses said nucleicacid or expresses a peptide, heterologous peptide, or protein encoded bya heterologous nucleic acid. Recombinant cells can express genes or genefragments in either the sense or antisense form that are not foundwithin the native (non-recombinant) form of the cell. Recombinant cellscan also express genes that are found in the native form of the cell,but wherein the genes are modified and re-introduced into the cell byartificial means.

A “structural gene” is that portion of a gene comprising a DNA segmentencoding a protein, polypeptide or a portion thereof, and excluding the5′ sequence which drives the initiation of transcription. The structuralgene may alternatively encode a nontranslatable product. The structuralgene may be one which is normally found in the cell or one which is notnormally found in the cell or cellular location wherein it isintroduced, in which case it is termed a “heterologous gene”. Aheterologous gene may be derived in whole or in part from any sourceknown to the art, including a bacterial genome or episome, eukaryotic,nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesized DNA. Astructural gene may contain one or more modifications that could effectbiological activity or its characteristics, the biological activity orthe chemical structure of the expression product, the rate of expressionor the manner of expression control. Such modifications include, but arenot limited to, mutations, insertions, deletions and substitutions ofone or more nucleotides. The structural gene may constitute anuninterrupted coding sequence or it may include one or more introns,bounded by the appropriate splice junctions. The structural gene may betranslatable or non-translatable, including in an anti-senseorientation. The structural gene may be a composite of segments derivedfrom a plurality of sources and from a plurality of gene sequences(naturally occurring or synthetic, where synthetic refers to DNA that ischemically synthesized).

“Derived from” is used to mean taken, obtained, received, traced,replicated or descended from a source (chemical and/or biological). Aderivative may be produced by chemical or biological manipulation(including, but not limited to, substitution, addition, insertion,deletion, extraction, isolation, mutation and replication) of theoriginal source.

“Chemically synthesized”, as related to a sequence of DNA, means thatportions of the component nucleotides were assembled in vitro. Manualchemical synthesis of DNA may be accomplished using well establishedprocedures (Caruthers, Methodology of DNA and RNA Sequencing, (1983),Weissman (ed.), Praeger Publishers, New York, Chapter 1); automatedchemical synthesis can be performed using one of a number ofcommercially available machines.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman and Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.,1990) is available from several sources, including the National Centerfor Biological Information (NCBI, Bethesda, Md.) and on the Internet,for use in connection with the sequence analysis programs blastp,blastn, blastx, tblastn and tblastx. It can be accessed athtp://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determinesequence identity using this program is available athttp://www.ncbi.nlm.nih.gov/BLAST/blast help.html.

The terms “substantial amino acid identity” or “substantial amino acidsequence identity” as applied to amino acid sequences and as used hereindenote a characteristic of a polypeptide, wherein the peptide comprisesa sequence that has at least 70 percent sequence identity, preferably 80percent amino acid sequence identity, more preferably 90 percent aminoacid sequence identity, and most preferably at least 99 to 100 percentsequence identity as compared to a reference group over regioncorresponding to the first amino acid following the cytochrome p450motif GXRXCX(G/A) to the stop codon of the translated peptide.

The terms “substantial nucleic acid identity” or “substantial nucleicacid sequence identity” as applied to nucleic acid sequences and as usedherein denote a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 75 percentsequence identity, preferably 81 percent sequence identity, morepreferably at least 91 percent sequence identity, and most preferably atleast 99 to 100 percent sequence identity as compared to a referencegroup over region corresponding to the first nucleic acid following thecytochrome p450 motif GXRXCX(G/A) to the stop codon of the translatedpeptide.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. to about 20° C., usually about 10° C. to about 15° C.,lower than the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength and pH) at which 50% of the target sequence hybridizes toa matched probe. Typically, stringent conditions will be those in whichthe salt concentration is about 0.02 molar at pH 7 and the temperatureis at least about 60° C. For instance in a standard Southernhybridization procedure, stringent conditions will include an initialwash in 6×SSC at 42° C. followed by one or more additional washes in0.2×SSC at a temperature of at least about 55° C., typically about 60°C. and often about 65° C.

Nucleotide sequences are also substantially identical for purposes ofthis invention when the polypeptides and/or proteins which they encodeare substantially identical. Thus, where one nucleic acid sequenceencodes essentially the same polypeptide as a second nucleic acidsequence, the two nucleic acid sequences are substantially identical,even if they would not hybridize under stringent conditions due todegeneracy permitted by the genetic code (see, Darnell et al. (1990)Molecular Cell Biology, Second Edition Scientific American Books W. H.Freeman and Company New York for an explanation of codon degeneracy andthe genetic code). Protein purity or homogeneity can be indicated by anumber of means well known in the art, such as polyacrylamide gelelectrophoresis of a protein sample, followed by visualization uponstaining. For certain purposes high resolution may be needed and HPLC ora similar means for purification may be utilized.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) into a cell. A vector may act toreplicate DNA and may reproduce independently in a host cell. The term“vehicle” is sometimes used interchangeably with “vector.” The term“expression vector” as used herein refers to a recombinant DNA moleculecontaining a desired coding sequence and appropriate nucleic acidsequences necessary for the expression of the operably linked codingsequence in a particular host organism. Nucleic acid sequences necessaryfor expression in prokaryotes usually include a promoter, an operator(optional), and a ribosome binding site, often along with othersequences. Eucaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

For the purpose of regenerating complete genetically engineered plantswith roots, a nucleic acid may be inserted into plant cells, forexample, by any technique such as in vivo inoculation or by any of theknown in vitro tissue culture techniques to produce transformed plantcells that can be regenerated into complete plants. Thus, for example,the insertion into plant cells may be by in vitro inoculation bypathogenic or non-pathogenic A. tumefaciens. Other such tissue culturetechniques may also be employed.

“Plant tissue” includes differentiated and undifferentiated tissues ofplants, including, but not limited to, roots, shoots, leaves, pollen,seeds, tumor tissue and various forms of cells in culture, such assingle cells, protoplasts, embryos and callus tissue. The plant tissuemay be in planta or in organ, tissue or cell culture.

“Plant cell” as used herein includes plant cells in planta and plantcells and protoplasts in culture.

“cDNA” or “complementary DNA” generally refers to a single stranded DNAmolecule with a nucleotide sequence that is complementary to an RNAmolecule. cDNA is formed by the action of the enzyme reversetranscriptase on an RNA template.

Strategies for Obtaining Nucleic Acid Sequences

In accordance with the present invention, RNA was extracted fromNicotiana tissue of converter and non-converter Nicotiana lines. Theextracted RNA was then used to create cDNA. Nucleic acid sequences ofthe present invention were then generated using two strategies.

In the first strategy, the poly A enriched RNA was extracted from planttissue and cDNA was made by reverse transcription PCR. The single strandcDNA was then used to create p450 specific PCR populations usingdegenerate primers plus a oligo d(T) reverse primer. The primer designwas based on the highly conserved motifs of p450. Examples of specificdegenerate primers are set forth in FIG. 1. Sequence fragments fromplasmids containing appropriate size inserts were further analyzed.These size inserts typically ranged from about 300 to about 800nucleotides depending on which primers were used.

In a second strategy, a cDNA library was initially constructed. The cDNAin the plasmids was used to create p450 specific PCR populations usingdegenerate primers plus T7 primer on plasmid as reverse primer. As inthe first strategy, sequence fragments from plasmids containingappropriate size inserts were further analyzed.

Nicotiana plant lines known to produce high levels of nornicotine(converter) and plant lines having undetectable levels of nornicotinemay be used as starting materials.

Leaves can then be removed from plants and treated with ethylene toactivate p450 enzymatic activities defined herein. Total RNA isextracted using techniques known in the art. cDNA fragments can then begenerated using PCR (RT-PCR) with the oligo d(T) primer as described inFIG. 153. The cDNA library can then be constructed more fully describedin examples herein.

The conserved region of p450 type enzymes can be used as a template fordegenerate primers (FIG. 75). Using degenerate primers, p450 specificbands can be amplified by PCR. Bands indicative for p450 like enzymescan be identified by DNA sequencing. PCR fragments can be characterizedusing BLAST search, alignment or other tools to identify appropriatecandidates.

Sequence information from identified fragments can be used to developPCR primers. These primers in combination of plasmid primers in cDNAlibrary were used to clone full length p450 genes. Large-scale Southernreverse analysis was conducted to examine the differential expressionfor all fragment clones obtained and in some cases full length clones.In this aspect of the invention, these large-scale reverse Southernassays can be conducted using labeled total cDNA's from differenttissues as a probe to hybridize with cloned DNA fragments in order toscreen all cloned inserts.

Nonradioactive and radioactive (P³²) Northern blotting assays were alsoused to characterize clones p450 fragments and full length clones.

Peptide specific antibodies were made against several full-length clonesby deriving their amino acid sequence and selecting peptide regions thatwere antigenic and unique relative to other clones. Rabbit antibodieswere made to synthetic peptides conjugated to a carrier protein. Westernblotting analyses or other immunological methods were performed on planttissue using these antibodies.

Nucleic acid sequences identified as described above can be examined byusing virus induced gene silencing technology (VIGS, Baulcombe, CurrentOpinions in Plant Biology, 1999, 2:109-113).

Peptide specific antibodies were made for several full-length clones byderiving their amino acid sequence and selecting peptide regions thatwere potentially antigenic and were unique relative to other clones.Rabbit antibodies were made to synthetic petides conjugated to a carrierprotein. Western blotting analyses were perfomed using these antibodies.

In another aspect of the invention, interfering RNA technology (RNAi) isused to further characterize cytochrome p450 enzymatic activities inNicotiana plants of the present invention. The following referenceswhich describe this technology are incorporated by reference herein,Smith et al., Nature, 2000, 407:319-320; Fire et al., Nature, 1998,391:306-311; Waterhouse et al., PNAS, 1998, 95:13959-13964; Stalberg etal., Plant Molecular Biology, 1993, 23:671-683; Baulcombe, CurrentOpinions in Plant Biology, 1999, 2:109-113; and Brigneti et al., EMBOJournal, 1998, 17(22):6739-6746. Plants may be transformed using RNAitechniques, antisense techniques, or a variety of other methodsdescribed.

Several techniques exist for introducing foreign genetic material intoplant cells, and for obtaining plants that stably maintain and expressthe introduced gene. Such techniques include acceleration of geneticmaterial coated onto microparticles directly into cells (U.S. Pat. No.4,945,050 to Cornell and U.S. Pat. No. 5,141,131 to DowElanco). Plantsmay be transformed using Agrobacterium technology, see U.S. Pat. No.5,177,010 to University of Toledo, U.S. Pat. No. 5,104,310 to Texas A&M,European Patent Application 0131624B1, European Patent Applications120516, 159418B1, European Patent Applications 120516, 159418B1 and176,112 to Schilperoot, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications116718, 290799, 320500 all to MaxPlanck, European Patent Applications604662 and 627752 to Japan Nicotiana, European Patent Applications0267159, and 0292435 and U.S. Pat. No. 5,231,019 all to Ciba Geigy, U.S.Pat. Nos. 5,463,174 and 4,762,785 both to Calgene, and U.S. Pat. Nos.5,004,863 and 5,159,135 both to Agracetus. Other transformationtechnology includes whiskers technology, see U.S. Pat. Nos. 5,302,523and 5,464,765 both to Zeneca. Electroporation technology has also beenused to transform plants, see WO 87/06614 to Boyce Thompson Institute,U.S. Pat. Nos. 5,472,869 and 5,384,253 both to Dekalb, WO9209696 andWO9321335 both to PGS. All of these transformation patents andpublications are incorporated by reference. In addition to numeroustechnologies for transforming plants, the type of tissue which iscontacted with the foreign genes may vary as well. Such tissue wouldinclude but would not be limited to embryogenic tissue, callus tissuetype I and II, hypocotyl, meristem, and the like. Almost all planttissues may be transformed during dedifferentiation using appropriatetechniques within the skill of an artisan.

Foreign genetic material introduced into a plant may include aselectable marker. The preference for a particular marker is at thediscretion of the artisan, but any of the following selectable markersmay be used along with any other gene not listed herein which couldfunction as a selectable marker. Such selectable markers include but arenot limited to aminoglycoside phosphotransferase gene of transposon Tn5(Aph II) which encodes resistance to the antibiotics kanamycin, neomycinand G418, as well as those genes which code for resistance or toleranceto glyphosate; hygromycin; methotrexate; phosphinothricin (bar);imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such aschlorosulfuron; bromoxynil, dalapon and the like.

In addition to a selectable marker, it may be desirous to use a reportergene. In some instances a reporter gene may be used without a selectablemarker. Reporter genes are genes which are typically not present orexpressed in the recipient organism or tissue. The reporter genetypically encodes for a protein which provide for some phenotypic changeor enzymatic property. Examples of such genes are provided in K. Weisinget al. Ann. Rev. Genetics, 22, 421 (1988), which is incorporated hereinby reference. Preferred reporter genes include without limitationglucuronidase (GUS) gene and GFP genes.

Once introduced into the plant tissue, the expression of the structuralgene may be assayed by any means known to the art, and expression may bemeasured as mRNA transcribed, protein synthesized, or the amount of genesilencing that occurs (see U.S. Pat. No. 5,583,021 which is herebyincorporated by reference). Techniques are known for the in vitroculture of plant tissue, and in a number of cases, for regeneration intowhole plants (EP Appln No. 88810309.0). Procedures for transferring theintroduced expression complex to commercially useful cultivars are knownto those skilled in the art.

Once plant cells expressing the desired level of p450 enzyme areobtained, plant tissues and whole plants can be regenerated therefromusing methods and techniques well-known in the art. The regeneratedplants are then reproduced by conventional means and the introducedgenes can be transferred to other strains and cultivars by conventionalplant breeding techniques.

The following examples illustrate methods for carrying out the inventionand should be understood to be illustrative of, but not limiting upon,the scope of the invention which is defined in the appended claims.

EXAMPLES Example I Development of Plant Tissue and Ethylene Treatment

Plant Growth

Plants were seeded in pots and grown in a greenhouse for 4 weeks. The 4week old seedlings were transplanted into individual pots and grown inthe greenhouse for 2 months. The plants were watered 2 times a day withwater containing 150 ppm NPK fertilizer during growth. The expandedgreen leaves were detached from plants to do the ethylene treatmentdescribed below.

Cell Line 78379

Tobacco line 78379, which is a burley tobacco line released by theUniversity of Kentucky was used as a source of plant material. Onehundred plants were cultured as standard in the art of growing tobaccoand transplanted and tagged with a distinctive number (1-100).Fertilization and field management were conducted as recommended.

Three quarters of the 100 plants converted between 20 and 100% of thenicotine to nornicotine. One quarter of the 100 plants converted lessthan 5% of the nicotine to nornicotine. Plant number 87 had the leastconversion (2%) while plant number 21 had 100% conversion. Plantsconverting less than 3% were classified as non-converters.Self-pollinated seed of plant number 87 and plant number 21, as well ascrossed (21×87 and 87×21) seeds were made to study genetic andphenotypic differences. Plants from selfed 21 were converters, and 99%of selfs from 87 were non-converters. The other 1% of the plants from 87showed low conversion (5-15%). Plants from reciprocal crosses were allconverters.

Cell Line 4407

Nicotiana line 4407, which is a burley line was used as a source ofplant material. Uniform and representative plants (100) were selectedand tagged. Of the 100 plants 97 were non-converters and three wereconverters. Plant number 56 had the least amount of conversion (1.2%)and plant number 58 had the highest level of conversion (96%).Self-pollenated seeds and crossed seeds were made with these two plants.

Plants from selfed-58 segregated with 3:1 converter to non-converterratio. Plants 58-33 and 58-25, were identified as homozygous converterand nonconverter plant lines, respectively. The stable conversion of58-33 was confirmed by analysis of its progenies of next generation.

Cell Line PBLB01

PBLB01 is a burley line developed by ProfiGen, Inc. and was used as asource of plant material. The converter plant was selected fromfoundation seeds of PBLB01.

Ethylene Treatment Procedures

Green leaves were detached from 2-3 month greenhouse grown plants andsprayed with 0.3% ethylene solution (Prep brand Ethephon(Rhone-Poulenc)). Each sprayed leaf was hung in a curing rack equippedwith humidifier and covered with plastic. During the treatment, thesample leaves were periodically sprayed with the ethylene solution.Approximately 24-48 hour post ethylene treatment, leaves were collectedfor RNA extraction. Another sub-sample was taken for metabolicconstituent analysis to determine the concentration of leaf metabolitesand more specific constituents of interest such as a variety ofalkaloids.

As an example, alkaloids analysis could be performed as follows. Samples(0.1 g) were shaken at 150 rpm with 0.5 ml 2N NaOH, and a 5 mlextraction solution which contained quinoline as an internal standardand methyl t-butyl ether. Samples were analyzed on a HP 6890 GC equippedwith a FID detector. A temperature of 250° C. was used for the detectorand injector. An HP column (30 m-0.32 nm-1·m) consisting of fused silicacrosslinked with 5% phenol and 95% methyl silicon was used at atemperature gradient of 110-185° C. at 10° C. per minute. The column wasoperated at 100° C. with a flow rate of 1.7 cm³ min⁻¹ with a split ratioof 40:1 with a 21 injection volume using helium as the carrier gas.

Example 2 RNA Isolation

For RNA extractions, middle leaves from 2 month old greenhouse grownplants were treated with ethylene as described. The 0 and 24-48 hourssamples were used for RNA extraction. In some cases, leaf samples underthe senescence process were taken from the plants 10 days postflower-head removal. These samples were also used for extraction. TotalRNA was isolated using Rneasy Plant Mini Kit® (Qiagen, Inc., Valencia,Calif.) following manufacturer's protocol.

The tissue sample was ground under liquid nitrogen to a fine powderusing a DEPC treated mortar and pestle. Approximately 100 milligrams ofground tissue were transferred to a sterile 1.5 ml eppendorf tube. Thissample tube was placed in liquid nitrogen until all samples werecollected. Then, 450μ-l of Buffer RLT as provided in the kit (with theaddition of Mercaptoethanol) was added to each individual tube. Thesample was vortexed vigorously and incubated at 56° C. for 3 minutes.The lysate was then, applied to the QIAshredder™ spin column sitting ina 2-ml collection tube, and centrifuged for 2 minutes at maximum speed.The flow through was collected and 0.5 volume of ethanol was added tothe cleared lysate. The sample is mixed well and transferred to anRneasy® mini spin column sitting in a 2 ml collection tube. The samplewas centrifuged for 1 minute at 10,000 rpm. Next, 700 μl of buffer RW1was pipetted onto the Rneasy® column and centrifuged for 1 minute at10,000 rpm. Buffer RPE was pipetted onto the Rneasy® column in a newcollection tube and centrifuged for 1 minute at 10,000 rpm. Buffer RPEwas again, added to the Rneasy® spin column and centrifuged for 2minutes at maximum speed to dry the membrane. To eliminate any ethanolcarry over, the membrane was placed in a separate collection tube andcentrifuged for an additional 1 minute at maximum speed. The Rneasy®column was transferred into a new 1.5 ml collection tube, and 40 μl ofRnase-free water was pipetted directly onto the Rneasy® membrane. Thisfinal elute tube was centrifuged for 1 minute at 10,000 rpm. Quality andquantity of total RNA was analyzed by denatured formaldehyde gel andspectrophotometer.

Poly(A)RNA was isolated using Oligotex™ poly A+ RNA purification kit(Qiagen Inc.) following manufacture's protocol. About 200 μg total RNAin 250 μl maximum volume was used. A volume of 250 μl of Buffer OBB and15 μl of Oligotex™ suspension was added to the 250 μl of total RNA. Thecontents were mixed thoroughly by pipetting and incubated for 3 minutesat 70° C. on a heating block. The sample was then, placed at roomtemperature for approximately 20 minutes. The oligotex:mRNA complex waspelleted by centrifugation for 2 minutes at maximum speed. All but 50 μlof the supernatant was removed from the microcentrifuge tube. The samplewas treated further by OBB buffer. The oligotex:mRNA pellet wasresuspended in 400 μl of Buffer OW2 by vortexing. This mix wastransferred onto a small spin column placed in a new tube andcentrifuged for 1 minute at maximum speed. The spin column wastransferred to a new tube and an additional 400 μl of Buffer OW2 wasadded to the column. The tube was then centrifuged for 1 minute atmaximum speed. The spin column was transferred to a final 1.5 mlmicrocentrifuge tube. The sample was eluted with 60 ul of hot (70° C.)Buffer OEB. Poly A product was analyzed by denatured formaldehyde gelsand spectrophotometric analysis.

Example 3 Reverse Transcription-PCR

First strand cDNA was produced using SuperScript reverse transcriptasefollowing manufacturer's protocol (Invitrogen, Carlsbad, Calif.). Thepoly A+ enriched RNA/oligo dT primer mix consisted of less than 5 μg oftotal RNA, 1 μl of 10 mM DNTP mix, 1 μl of Oligo d(T)₁₂₋₁₈ (0.5 μg/μl),and up to 10 μl of DEPC-treated water. Each sample was incubated at 65°C. for 5 minutes, then placed on ice for at least 1 minute. A reactionmixture was prepared by adding each of the following components inorder: 2 μl 10×RT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, and 1μl of RNase OUT Recombinant RNase Inhibitor. An addition of 9 μl ofreaction mixture was pipetted to each RNA/primer mixture and gentlymixed. It was incubated at 42° C. for 2 minutes and 1 μl of Super ScriptII™ RT was added to each tube. The tube was incubated for 50 minutes at42° C. The reaction was terminated at 70° C. for 15 minutes and chilledon ice. The sample was collected by centrifugation and 1 μl of RNase Hwas added to each tube and incubated for 20 minutes at 37° C. The secondPCR was carried out with 200 pmoles of forward primer (degenerateprimers as in FIG. 75, SEQ. ID Nos. 149-156) and 100 pmoles reverseprimer (mix of 18 nt oligo d(T) followed by 1 random base).

Reaction conditions were 94° C. for 2 minutes and then performed 40cycles of PCR at 94° C. for 1 minute, 45° to 60° C. for 2 minutes, 72°C. for 3 minutes with a 72° C. extension for an extra 10 min.

Ten microliters of the amplified sample were analyzed by electrophoresisusing a 1% agarose gel. The correct size fragments were purified fromagarose gel.

Example 4 Generation of PCR Fragment Populations

PCR fragments from Example 3 were ligated into a pGEM-T® Easy Vector(Promega, Madison, Wis.) following manufacturer's instructions. Theligated product was transformed into JM109 competent cells and plated onLB media plates for blue/white selection. Colonies were selected andgrown in a 96 well plate with 1.2 ml of LB media overnight at 37° C.Frozen stock was generated for all selected colonies. Plasmid DNA fromplates were purified using Beckman's Biomeck 2000 miniprep robotics withWizard SV Miniprep® kit (Promega). Plasmid DNA was eluted with 100 μlwater and stored in a 96 well plate. Plasmids were digested by EcoR1 andwere analyzed using 1% agarose gel to confirm the DNA quantity and sizeof inserts. The plasmids containing a 400-600 bp insert were sequencedusing an CEQ 2000 sequencer (Beckman, Fullerton, Calif.). The sequenceswere aligned with GenBank database by BLAST search. The p450 relatedfragments were identified and further analyzed. Alternatively, p450fragments were isolated from substraction libraries. These fragmentswere also analyzed as described above.

Example 5 Construction of cDNA library

A cDNA library was constructed by preparing total RNA from ethylenetreated leaves as follows. First, total RNA was extracted from ethylenetreated leaves of tobacco line 58-33 using a modified acid phenol andchloroform extraction protocol. Protocol was modified to use one gram oftissue that was ground and subsequently vortexed in 5 ml of extractionbuffer (100 mM Tris-HCl, pH 8.5; 200 mM NaCl; 10 mM EDTA; 0.5% SDS) towhich 5 ml phenol (pH5.5) and 5 ml chloroform was added. The extractedsample was centrifuged and the supernatant was saved. This extractionstep was repeated 2-3 more times until the supernatant appeared clear.Approximately 5 ml of chloroform was added to remove trace amounts ofphenol. RNA was precipitated from the combined supernatant fractions byadding a 3-fold volume of ETOH and 1/10 volume of 3M NaOAc (pH5.2) andstoring at −20° C. for 1 hour. After transferring to a Corex glasscontainer the RNA fraction was centrifuged at 9,000 RPM for 45 minutesat 4° C. The pellet was washed with 70% ethanol and spun for 5 minutesat 9,000 RPM at 4° C. After drying the pellet, the pelleted RNA wasdissolved in 0.5 ml RNase free water. The pelleted RNA was dissolved in0.5 ml RNase free water. The quality and quantity of total RNA wasanalyzed by denatured formaldehyde gel and spectrophotometer,respectively.

The resultant total RNA was isolated for poly A+ RNA using an Oligo(dT)cellulose protocol (Invitrogen) and Microcentrifuge spin columns(Invitrogen) by the following protocol. Approximately twenty mg of totalRNA was subjected to twice purification to obtain high quality poly A+RNA. Poly A+ RNA product was analyzed by performing denaturedformaldehyde gel and subsequent RT-PCR of known full-length genes toensure high quality of mRNA.

Next, poly A+ RNA was used as template to produce a cDNA libraryemploying cDNA synthesis kit, ZAP-cDNA® synthesis kit, and ZAP-cDNA®Gigapack® III gold cloning kit (Stratagene, La Jolla, Calif.). Themethod involved following the manufacture's protocol as specified.Approximately 8 μg of poly A+ RNA was used to construct cDNA library.Analysis of the primary library revealed about 2.5×10⁶-1×10⁷ pfu. Aquality background test of the library was completed by complementationassays using IPTG and X-gal, where recombinant plaques was expressed atmore than 100-fold above the background reaction.

A more quantitative analysis of the library by random PCR showed thataverage size of insert cDNA was approximately 1.2 kb. The method used atwo-step PCR method as followed. For the first step, reverse primerswere designed based on the preliminary sequence information obtainedfrom p450 fragments. The designed reverse primers and T3 (forward)primers were used amplify corresponding genes from the cDNA library. PCRreactions were subjected to agarose electrophoresis and thecorresponding bands of high molecular weight were excised, purified,cloned and sequenced. In the second step, new primers designed from5′UTR or the start coding region of p450 as the forward primers togetherwith the reverse primers (designed from 3′UTR of p450) were used in thesubsequent PCR to obtain full-length p450 clones.

The p450 fragments were generated by PCR amplification from theconstructed cDNA library as described in Example 3 with the exception ofthe reverse primer. The T7 primer located on the plasmid downstream ofcDNA inserts (see FIG. 75) was used as a reverse primer. PCR fragmentswere isolated, cloned and sequenced as described in Example 4.

Full-length p450 genes were isolated by PCR method from constructed cDNAlibrary. Gene specific reverse primers (designed from the downstreamsequence of p450 fragments) and a forward primer (T3 on library plasmid)were used to clone the full length genes. PCR fragments were isolated,cloned and sequenced. If necessary, second step PCR was applied. In thesecond step, new forward primers designed from 5′UTR of cloned p450stogether with the reverse primers designed from 3′UTR of p450 cloneswere used in the subsequent PCR reactions to obtain full-length p450clones. The clones were subsequently sequenced.

Example 6 Characterization of Cloned Fragments—Reverse Southern BlottingAnalysis

Nonradioactive large scale reverse southern blotting assays wereperformed on all p450 clones identified in above examples to detect thedifferential expression. It was observed that the level of expressionamong different p450 clusters was very different. Further real timedetection was conducted on those with high expression.

Nonradioactive Southern blotting procedures were conducted as follows.

1) Total RNA was extracted from ethylene treated and nontreatedconverter (58-33) and nonconverter (58-25) leaves using the QiagenRnaeasy kit as described in Example 2.

2) Probe was produced by biotin-tail labeling a single strand cDNAderived from poly A+ enriched RNA generated in above step. This labeledsingle strand cDNA was generated by RT-PCR of the converter andnonconverter total RNA (Invitrogen) as described in Example 3 with theexception of using biotinalyted oligo dT as a primer (Promega). Thesewere used as a probe to hybridize with cloned DNA.

3) Plasmid DNA was digested with restriction enzyme EcoR1 and run onagarose gels. Gels were simultaneously dried and transferred to twonylon membranes (Biodyne B®). One membrane was hybridized with converterprobe and the other with nonconverter probe. Membranes wereUV-crosslinked (auto crosslink setting, 254 nm, Stratagene,Stratalinker) before hybridization.

Alternatively, the inserts were PCR amplified from each plasmid usingthe sequences located on both arms of p-GEM plasmid, T3 and SP6, asprimers. The PCR products were analyzed by running on a 96 wellReady-to-run agarose gels. The confirmed inserts were dotted on twonylon membranes. One membrane was hybridized with converter probe andthe other with nonconverter probe.

4) The membranes were hybridized and washed following manufacture'sinstruction with the modification of washing stringency (Enzo MaxSence™kit, Enzo Diagnostics, Inc, Farmingdale, N.Y.). The membranes wereprehybridized with hybridization buffer (2×SSC buffered formamide,containing detergent and hybridization enhancers) at 42° C. for 30 minand hybridized with 101 denatured probe overnight at 42° C. Themembranes then were washed in 1× hybridization wash buffer 1 time atroom temperature for 10 min and 4 times at 68° C. for 15 min. Themembranes were ready for the detection.

5) The washed membranes were detected by alkaline phosphatase labelingfollowed by NBT/BCIP colometric detection as described in manufacture'sdetection procedure (Enzo Diagnostics, Inc.). The membranes were blockedfor one hour at room temperature with 1× blocking solution, washed 3times with 1× detection reagents for 10 min, washed 2 times with 1×predevelopment reaction buffer for 5 min and then developed the blots indeveloping solution for 30-45 min until the dots appear. All reagentswere provided by manufacture (Enzo Diagnostics, Inc). In Addition, largescale reverse Southern assay was also performed using KPL southernhybridization and detection kit™ following manfacturer's instruction(KPL, Gaithersburg, Md.).

Example 7 Characterization of Clones—Northern Blot Analysis

Alternative to Southern Blot analysis, some membranes were hybridizedand detected as described in the example of Northern blotting assays.Northern Hybridization was used to detect mRNA differentially expressedin Nicotiana as follows.

A random priming method was used to prepare probes from cloned p450(Megaprime™ DNA Labelling Systems, Amersham Biosciences).

The following components were mixed: 25 ng denatured DNA template; 4 ulof each unlabeled dTTP, dGTP and dCTP; 5 ul of reaction buffer;P³²-labelled dATP and 2 ul of Klenow I; and H₂O, to bring the reactionto 50 μl. The mixture was incubated in 37° C. for 1-4 hours, thenstopped with 2 μl of 0.5 M EDTA. The probe was denatured by incubatingat 95° C. for 5 minutes before use.

RNA samples were prepared from ethylene treated and non-treated freshleaves of several pairs of tobacco lines. In some cases poly A+ enrichedRNA was used. Approximately 15 μg total RNA or 1.8 μg mRNA (methods ofRNA and mRNA extraction as described in Example 5) were brought to equalvolume with DEPC H₂O (5-10 μl). The same volume of loading buffer(1×MOPS; 18.5% Formaldehyde; 50% Formamide; 4% Ficoll400;Bromophenolblue) and 0.5 μl EtBr (0.5 μg/μl) were added. The sampleswere subsequently denatured in preparation for separation of the RNA byelectrophoresis.

Samples were subjected to electrophoresis on a formaldehyde gel (1%Agarose, 1×MOPS, 0.6 M Formaldehyde) with 1×MOP buffer (0.4 MMorpholinopropanesulfonic acid; 0.1 M Na-acetate-3×H2O; 10 mM EDTA;adjust to pH 7.2 with NaOH). RNA was transferred to a Hybond-N+ membrane(Nylon, Amersham Pharmacia Biotech) by capillary method in 10×SSC buffer(1.5 M NaCl; 0.15 M Na-citrate) for 24 hours. Membranes with RNA sampleswere UV-crosslinked (auto crosslink setting, 254 nm, Stratagene,Stratalinker) before hybridization.

The membrane was prehybridized for 1-4 hours at 42° C. with 5-10 mlprehybridization buffer (5×SSC; 50% Formamide; 5× Denhardt's-solution;1% SDS; 100 μg/ml heat-denatured sheared non-homologous DNA). Oldprehybridization buffer was discarded, and new prehybridization bufferand probe were added. The hybridization was carried out over night at42° C. The membrane was washed for 15 minutes with 2×SSC at roomtemperature, followed by a wash with 2×SSC.

A major focus of the invention was the discovery of novel genes that maybe induced as a result of ethylene treatment or play a key role intobacco leaf quality and constituents. As illustrated in the tablebelow, Northern blots and reverse Southern Blot were useful indetermining which genes were induced by ethylene treatment relative tonon-induced plants. Interestingly, not all fragments were affectedsimilarly in the converter and nonconverter. The cytochrome p450fragments of interest were partially sequenced to determine theirstructural relatedness. This information was used to subsequentlyisolate and characterize full length gene clones of interest. InducedmRNA Expression Ethylene Treatment Fragments Converter D56-AC7 (SEQ IDNo: 35) + D56-AG11 (SEQ ID No: 31) + D56-AC12 (SEQ ID No: 45) + D70A-AB5(SEQ ID No: 95) + D73-AC9 (SEQ ID No: 43) + D70A-AA12 (SEQ ID No: 131) +D73A-AG3 (SEQ ID No: 129) + D34-52 (SEQ ID No: 61) + D56-AG6 (SEQ ID No:51) +

Northern analysis was performed using full length clones on tobaccotissue obtained from converter and nonconverter burley lines that wereinduced by ethylene treatment. The purpose was to identify those fulllength clones that showed elevated expression in ethylene inducedconverter lines relative to ethylene induced converter lines relative toethylene induced nonconverter burley lines. By so doing, thefunctionality relationship of full length clones may be determined bycomparing biochemical differences in leaf constituents between converterand nonconverter lines. As shown in table below, six clones showedsignificantly higher expression, as denoted by ++ and +++, in converterethylene treated tissue than that of nonconverter treated tissue,denoted by +. All of these clones showed little or no expression inconverter and nonconverter lines that were not ethylene treated. FullLength Clones Converter Nonconverter D101-BA2 ++ + D207-AA5 ++ +D208-AC8 +++ + D237-AD1 ++ + D89-AB1 ++ + D90A-BB3 ++ +

Example 8 Immunodetection of p450s Encoded by the Cloned Genes

Peptide regions corresponding to 20-22 amino acids in length from threep450 clones were selected for 1) having lower or no homology to otherclones and 2) having good hydrophilicity and antigenicity. The aminoacid sequences of the peptide regions selected from the respective p450clones are listed below. The synthesized peptides were conjugated withKHL and then injected into rabbits. Antisera were collected 2 and 4weeks after the 4^(th) injection (Alpha Diagnostic Intl. Inc. SanAntonio, Tex.). D234-AD1 DIDGSKSKLVKAHRKIDEILG D90a-BB3RDAFREKETFDENDVEELNY D89 -AB1 FKNNGDEDRHFSQKLGDLADKY

Antisera were examined for crossreactivity to target proteins fromtobacco plant tissue by Western Blot analysis. Crude protein extractswere obtained from ethylene treated (0 to 40 hours) middle leaves ofconverter and nonconverter lines. Protein concentrations of the extractswere determined using RC DC Protein Assay Kit (BIO-RAD) following themanufacturer's protocol.

Two micrograms of protein were loaded onto each lane and the proteinsseparated on 10%-20% gradient gels using the Laemmli SDS-PAGE system.The proteins were transferred from gels to PROTRAN® NitrocelluloseTransfer Membranes (Schleicher & Schuell) with the Trans-Blot® Semi-Drycell (BIO-RAD). Target p450 proteins were detected and visualized withthe ECL Advance™ Western Blotting Detection Kit (Amersham Biosciences).Primary antibodies against the synthetic-KLH conjugates were made inrabbits. Secondary antibody against rabbit IgG, coupled with peroxidase,was purchased from Sigma. Both primary and secondary antibodies wereused at 1:1000 dilutions. Antibodies showed strong reactivity to asingle band on the Western Blots indicating that the antisera weremonospecific to the target peptide of interest. Antisera were alsocrossreactive with synthetic peptides conjuated to KLH.

Example 9 Nucleic Acid Identity and Structure Relatedness of IsolatedNucleic Acid Fragments

Over 100 cloned p450 fragments were sequenced in conjunction withNorthern blot analysis to determine their structural relatedness. Theapproach used utilized forward primers based either of two common p450motifs located near the carboxyl-terminus of the p450 genes. The forwardprimers corresponded to cytochrome p450 motifs FXPERF or GRRXCP(A/G) asdenoted in FIG. 1. The reverse primers used standard primers from eitherthe plasmid, SP6 or T7 located on both arms of pGEM™ plasmid, or a polyA tail. The protocol used is described below.

Spectrophotometry was used to estimate the concentration of startingdouble stranded DNA following the manufacturer's protocol (BeckmanCoulter). The template was diluted with water to the appropriateconcentration, denatured by heating at 95° C. for 2 minutes, andsubsequently placed on ice. The sequencing reaction was prepared on iceusing 0.5 to 10 μl of denatured DNA template, 2 μl of 1.6 pmole of theforward primer, 8 μl of DTCS Quick Start Master Mix and the total volumebrought to 20 μl with water. The thermocycling program consisted of 30cycles of the follow cycle: 96° C. for 20 seconds, 50° C. for 20seconds, and 60° C. for 4 minutes followed by holding at 4° C.

The sequence was stopped by adding 5 μl of stop buffer (equal volume of3M NaOAc and 100 mM EDTA and 1 μl of 20 mg/ml glycogen). The sample wasprecipitated with 60 μl of cold 95% ethanol and centrifuged at 6000 gfor 6 minutes. Ethanol was discarded. The pellet was 2 washes with 200μl of cold 70% ethanol. After the pellet was dry, 40 μl of SLS solutionwas added and the pellet was resuspended. A layer of mineral oil wasover laid. The sample was then, placed on the CEQ 8000 AutomatedSequencer for further analysis.

In order to verify nucleic acid sequences, nucleic acid sequence wasre-sequenced in both directions using forward primers to the FXPERF orGRRXCP(A/G) region of the p450 gene or reverse primers to either theplasmid or poly A tail. All sequencing was performed at least twice inboth directions.

The nucleic acid sequences of cytochrome p450 fragments were compared toeach other from the coding region corresponding to the first nucleicacid after the region encoding the GRRXCP(A/G) motif through to the stopcodon. This region was selected as an indicator of genetic diversityamong p450 proteins. A large number of genetically distinct p450 genes,in excess of 70 genes, were observed, similar to that of other plantspecies. Upon comparison of nucleic acid sequences, it was found thatthe genes could be placed into distinct sequences groups based on theirsequence identity. It was found that the best unique grouping of p450members was determined to be those sequences with 75% nucleic acididentity or greater (shown in Table I). Reducing the percentage identityresulted in significantly larger groups. A preferred grouping wasobserved for those sequences with 81% nucleic acid identity or greater,a more preferred grouping 91% nucleic acid identity or greater, and amost preferred grouping for those sequences 99% nucleic acid identity ofgreater. Most of the groups contained at least two members andfrequently three or more members. Others were not repeatedly discoveredsuggesting that approach taken was able to isolated both low and highexpressing mRNA in the tissue used.

Based on 75% nucleic acid identity or greater, two cytochrome p450groups were found to contain nucleic acid sequence identity topreviously tobacco cytochrome genes that genetically distinct from thatwithin the group. Group 23, showed nucleic acid identity, within theparameters used for Table I, to prior GenBank sequences of GI:1171579(CAA64635) and GI:14423327 (or AAK62346) by Czernic et al and Ralston etal, respectively. GI:1171579 had nucleic acid identity to Group 23members ranging 96.9% to 99.5% identity to members of Group 23 whileGI:14423327 ranged 95.4% to 96.9% identity to this group. The members ofGroup 31 had nucleic acid identity ranging from 76.7% to 97.8% identityto the GenBank reported sequence of GI:14423319 (AAK62342) by Ralston etal. None of the other p450 identity groups of Table 1 containedparameter identity, as used in Table 1, to Nicotiana p450s genesreported by Ralston et al, Czernic et al., Wang et al or LaRosa andSmigocki.

As shown in FIG. 76, consensus sequence with appropriate nucleic aciddegenerate probes could be derived for group to preferentially identifyand isolate additional members of each group from Nicotiana plants.TABLE I Nicotiana p450 Nucleic Acid Sequence Identity Groups GROUPFRAGMENTS 1 D58-BG7 (SEQ ID No.: 1), D58-AB1 (SEQ ID No.: 3); D58-BE4(SEQ ID No.: 7) 2 D56-AH7 (SEQ ID No.: 9); D13a-5 (SEQ ID No.: 11) 3D56-AG10 (SEQ ID No.: 13); D35-33 (SEQ ID No.: 15); D34-62 (SEQ ID No.:17) 4 D56-AA7 (SEQ ID No.: 19); D56-AE1 (SEQ ID No.: 21); 185-BD3 (SEQID No.: 143) 5 D35-BB7 (SEQ ID No.: 23); D177-BA7 (SEQ ID No.: 25);D56A-AB6 (SEQ ID No.: 27); D144-AE2 (SEQ ID No.: 29) 6 D56-AG11 (SEQ IDNo.: 31); D179-AA1 (SEQ ID No.: 33) 7 D56-AC7 (SEQ ID No.: 35); D144-AD1(SEQ ID No.: 37) 8 D144-AB5 (SEQ ID No.: 39) 9 D181-AB5 (SEQ ID No.:41); D73-Ac9 (SEQ ID No.: 43) 10 D56-AC12 (SEQ ID No.: 45) 11 D58-AB9(SEQ ID No.: 47); D56-AG9 (SEQ ID No.: 49); D56-AG6 (SEQ ID No.: 51);D35-BG11 (SEQ ID No.: 53); D35-42 (SEQ ID No.: 55); D35-BA3 (SEQ ID No.:57); D34-57 (SEQ ID No.: 59); D34-52 (SEQ ID No.: 61); D34-25 (SEQ IDNo.: 63) 12 D56-AD10 (SEQ ID No.: 65) 13 56-AA11 (SEQ ID No.: 67) 14D177-BD5 (SEQ ID No.: 69); D177-BD7 (SEQ ID No.: 83) 15 D56A-AG10 (SEQID No.: 71); D58-BC5 (SEQ ID No.: 73); D58-AD12 (SEQ ID No.: 75) 16D56-AC11 (SEQ ID No.: 77); D35-39 (SEQ ID No.: 79); D58-BH4 (SEQ ID No.:81); D56-AD6 (SEQ ID No.: 87) 17 D73A-AD6 (SEQ ID No.: 89); D70A-BA11(SEQ ID No.: 91) 18 D70A-AB5 (SEQ ID No.: 95); D70A-AA8 (SEQ ID No.: 97)19 D70A-AB8 (SEQ ID No.: 99); D70A-BH2 (SEQ ID No.: 101); D70A-AA4 (SEQID No.: 103) 20 D70A-BA1 (SEQ ID No.: 105); D70A-BA9 (SEQ ID No.: 107)21 D70A-BD4 (SEQ ID No.: 109) 22 D181-AC5 (SEQ ID No.: 111); D144-AH1(SEQ ID No.: 113); D34-65 (SEQ ID No.: 115) 23 D35-BG2 (SEQ ID No.: 117)24 D73A-AH7 (SEQ ID No.: 119) 25 D58-AA1 (SEQ ID No.: 121); D185-BC1(SEQ ID No.: 133); D185-BG2 (SEQ ID No.: 135) 26 D73-AE10 (SEQ ID No.:123) 27 D56-AC12 (SEQ ID No.: 125) 28 D177-BF7 (SEQ ID No.: 127);D185-BE1 (SEQ ID No.: 137); D185-BD2 (SEQ ID No.: 139) 29 D73A-AG3 (SEQID No.: 129) 30 D70A-AA12 (SEQ ID No.: 131); D176-BF2 (SEQ ID No.: 85)31 D176-BC3 (SEQ ID No.: 145) 32 D176-BB3 (SEQ ID No.: 147) 33 D186-AH4(SEQ ID No.: 5)

Example 10 Related Amino Acid Sequence Identity of Isolated Nucleic AcidFragments

The amino acid sequences of nucleic acid sequences obtained forcytochrome p450 fragments from Example 8 were deduced. The deducedregion corresponded to the amino acid immediately after the GXRXCP(A/G)sequence motif to the end of the carboxyl-terminus, or stop codon. Uponcomparison of sequence identity of the fragments, a unique grouping wasobserved for those sequences with 70% amino acid identity or greater. Apreferred grouping was observed for those sequences with 80% amino acididentity or greater, more preferred with 90% amino acid identity orgreater, and a most preferred grouping for those sequences 99% aminoacid identity of greater. The groups and corresponding amino acidsequences of group members are shown in FIG. 2. Several of the uniquenucleic acid sequences were found to have complete amino acid identityto other fragments and therefore only one member with the identicalamino acid was reported.

The amino acid identity for Group 19 of Table II corresponded to threedistinct groups based on their nucleic acid sequences. The amino acidsequences of each group member and their identity is shown in FIG. 77.The amino acid differences are appropriated marked.

At least one member of each amino acid identity group was selected forgene cloning and functional studies using plants. In addition, groupmembers that are differentially affected by ethylene treatment or otherbiological differences as assessed by Northern and Southern analysiswere selected for gene cloning and functional studies. To assist in genecloning, expression studies and whole plant evaluations, peptidespecific antibodies will be prepared on sequence identity anddifferential sequence. TABLE II Nicotiana p450 Amino Acid SequenceIdentity Groups GROUP FRAGMENTS 1 D58-BG7 (SEQ ID No.: 2), D58-AB1 (SEQID No.: 4) 2 D58-BE4 (SEQ ID No.: 8) 3 D56-AH7 (SEQ ID No.: 10); D13a-5(SEQ ID No.: 12) 4 D56-AG10 (SEQ ID No.: 14); D34-62 (SEQ ID No.: 18) 5D56-AA7 (SEQ ID No.: 20); D56-AE1 (SEQ ID No.: 22); 185- BD3 (SEQ IDNo.: 144) 6 D35-BB7 (SEQ ID No.: 24); D177-BA7 (SEQ ID No.: 26);D56A-AB6 (SEQ ID No.: 28); D144-AE2 (SEQ ID No.: 30) 7 D56-AG11 (SEQ IDNo.: 32); D179-AA1 (SEQ ID No.: 34) 8 D56-AC7 (SEQ ID No.: 36); D144-AD1(SEQ ID No.: 38) 9 D144-AB5 (SEQ ID No.: 40) 10 D181-AB5 (SEQ ID No.:42); D73-Ac9 (SEQ ID No.: 44) 11 D56-AC12 (SEQ ID No.: 46) 12 D58-AB9(SEQ ID No.: 48); D56-AG9 (SEQ ID No.: 50); D56-AG6 (SEQ ID No.: 52);D35-BG11 (SEQ ID No.: 54); D35-42 (SEQ ID No.: 56); D35-BA3 (SEQ ID No.:58); D34-57 (SEQ ID No.: 60); D34-52 (SEQ ID No.: 62) 13 D56AD10 (SEQ IDNo.: 66) 14 56-AA11 (SEQ ID No.: 68) 15 D177-BD5 (SEQ ID No.: 70);D177-BD7 (SEQ ID No.: 84) 16 D56A-AG10 (SEQ ID No.: 72); D58-BC5 (SEQ IDNo.: 74); D58-AD12 (SEQ ID No.: 76) 17 D56-AC11 (SEQ ID No.: 78);D56-AD6 (SEQ ID No.: 88) 18 D73A-AD6 (SEQ ID No. 90:) 19 D70A-AB5 (SEQID No.: 96); D70A-AB8 (SEQ ID No.: 100); D70A-BH2 (SEQ ID No.: 102);D70A-AA4 (SEQ ID No.: 104); D70A-BA1 (SEQ ID No.: 106); D70A-BA9 (SEQ IDNo.: 108) 20 D70A-BD4 (SEQ ID No.: 110) 21 D181-AC5 (SEQ ID No.: 112);D144-AH1 (SEQ ID No.: 114); D34-65 (SEQ ID No.: 116) 22 D35-BG2 (SEQ IDNo.: 118) 23 D73A-AH7 (SEQ ID No.: 120) 24 D58-AA1 (SEQ ID No.: 122);D185-BC1 (SEQ ID No.: 134); D185-BG2 (SEQ ID No.: 136) 25 D73-AE10 (SEQID No.: 124) 26 D56-AC12 (SEQ ID No.: 126) 27 D177-BF7 (SEQ ID No.:128); 185-BD2 (SEQ ID No.: 140) 28 D73A-AG3 (SEQ ID No.: 130) 29D70A-AA12 (SEQ ID No.: 132); D176-BF2 (SEQ ID No.: 86) 30 D176-BC3 (SEQID No.: 146) 31 D176-BB3 (SEQ ID No.: 148) 32 D186-AH4 (SEQ ID No.: 6)

Example 11 Related Amino Acid Sequence Identity of Full Length Clones

The nucleic acid sequence of full length Nicotiana genes cloned inExample 5 were deduced for their entire amino acid sequence. Cytochromep450 genes were identified by the presence of three conserved p450domain motifs, which corresponded to UXXRXXZ, PXRFXF or GXRXC at thecarboxyl-terminus where U is E or K, X is any amino acid and Z is P, T,S or M. It was also noted that two of the clones appeared nearlycomplete but lacked the appropriate stop codon, D130-AA1 and D101-BA2,however but both contained all three p450 cytochrome domains. All p450genes were characterized for amino acid identity using a BLAST programcomparing their full length sequences to each other and to known tobaccogenes. The program used the NCBI special BLAST tool (Align two sequences(b12seq), http://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html). Twosequences were aligned under BLASTN without filter for nucleic acidsequences and BLASTP for amino acid sequences. Based on their percentageamino acid identity, each sequence was grouped into identity groupswhere the grouping contained members that shared at least 85% identitywith another member. A preferred grouping was observed for thosesequences with 90% amino acid identity or greater, a more preferredgrouping had 95% amino acid identity or greater, and a most preferredgrouping had those sequences 99% amino acid identity or greater. Usingthese criteria, 25 unique groups were identified and are depicted inTable III.

Within the parameters used for Table III for amino acid identity, threegroups were found to contain greater than 85% or greater identity toknown tobacco genes. Members of Group 5 had up to 96% amino acididentity for full length sequences to prior GenBank sequences ofGI:14423327 (or AAK62346) by Ralston et al. Group 23 had up to 93% aminoacid identity to GI:14423328 (or AAK62347) by Ralston et al. and Group24 had 92% identity to GI:14423318 (or AAK62343) by Ralston et al. TABLEIII Amino Acid Sequence Identity Groups of Full Length Nicotiana p450Genes 1 D208-AD9 (SEQ. ID. No. 224); D120-AH4 (SEQ. ID. No. 180);D121-AA8 (SEQ. ID. No. 182) , D122-AF10 (SEQ. ID. No. 184); D103-AH3(SEQ. ID. No. 222); D208-AC8 (SEQ. ID. No. 218); D-235-ABI (SEQ. ID. No.246) 2 D244-AD4 (SEQ. ID. No. 250); D244-AB6 (SEQ. ID. No. 274);D285-AA8; D285-AB9; D268-AE2 (SEQ. ID. No. 270) 3 D100A-AC3 (SEQ. ID.No. 168); D100A-BE2 4 D205-BE9 (SEQ. ID. No. 276); D205-BG9 (SEQ. ID.No. 202); D205-AH4 (SEQ. ID. No. 294) 5 D259-AB9 (SEQ. ID. No. 260);D257-AE4 (SEQ. ID. No. 268); D147-AD3 (SEQ. ID. No. 194) 6 D249-AE8(SEQ. ID. No. 256); D-248-AA6 (SEQ. ID. No. 254) 7 D233-AG7 (SEQ. ID.No. 266; D224-BD11 (SEQ. ID. No. 240); DAF10 8 D105-AD6 (SEQ. ID. No.172); D215-AB5 (SEQ. ID. No. 220); D135-AE1 (SEQ. ID. No. 190) 9D87A-AF3 (SEQ. ID. No. 216), D210-BD4 (SEQ. ID. No. 262) 10 D89-AB1(SEQ. ID. No. 150); D89-AD2 (SEQ. ID. No. 152); 163-AG11 (SEQ. ID. No.198); 163-AF12 (SEQ. ID. No. 196) 11 D267-AF10 (SEQ. ID. No. 296);D96-AC2 (SEQ. ID. No. 160); D96-AB6 (SEQ. ID. No. 158); D207-AA5 (SEQ.ID. No. 204); D207-AB4 (SEQ. ID. No. 206); D207-AC4 (SEQ. ID. No. 208)12 D98-AG1 (SEQ. ID. No. 164); D98-AA1 (SEQ. ID. No. 162) 13 D209-AA12(SEQ. ID. No. 212); D209-AA11; D209-AH10 (SEQ. ID. No. 214); D209-AH12(SEQ. ID. No. 232); D90a-BB3 (SEQ. ID. No. 154) 14 D129-AD10 (SEQ. ID.No. 188); D104A-AE8 (SEQ. ID. No. 170) 15 D228-AH8 (SEQ. ID. No. 244);D228-AD7 (SEQ. ID. No. 241) , D250-AC11 (SEQ. ID. No. 258); D247-AH1(SEQ. ID. No. 252) 16 D128-AB7 (SEQ. ID. No. 186); D243-AA2 (SEQ. ID.No. 248); D125-AF11 (SEQ. ID. No. 228) 17 D284-AH5 (SEQ. ID. No. 298);D110-AF12 (SEQ. ID. No. 176) 18 D221-BB8 (SEQ. ID. No. 234) 19 D222-BH4(SEQ. ID. No. 236) 20 D134-AE11 (SEQ. ID. No. 230) 21 D109-AH8 (SEQ. ID.No. 174) 22 D136-AF4 (SEQ. ID. No. 278) 23 D237-AD1 (SEQ. ID. No. 226)24 D112-AA5 (SEQ. ID. No. 178) 25 D283-AC1 (SEQ. ID. No. 272)

The full length genes were further grouped based on the highly conversedamino acid homology between UXXRXXZ p450 domain and GXRXC p450 domainnear the end the carboxyl-terminus. As shown in FIG. 3, individualclones were aligned for their sequence homology between the conserveddomains relative to each other and placed in distinct identity groups.In several cases, although the nucleic acid sequence of the clone wasunique, the amino acid sequence for the region was identical. Thepreferred grouping was observed for those sequences with 90% amino acididentity or greater, a more preferred group had 95% amino acid identityor greater, and a most preferred grouping had those sequences 99% aminoacid identity of greater. The final grouping was similar to that basedon the percent identity for the entire amino acid sequence of the clonesexcept for Group 17 (of Table III) which was divided into two distinctgroups.

Within the parameters used for amino acid identity in Table IV, threegroups were found to contain 90% or greater identity to known tobaccogenes. Members of Group 5 had up to 93.4% amino acid identity for fulllength sequences to prior GenBank sequences of GI:14423326 (AAK62346) byRalston et al. Group 23 had up to 91.8% amino acid identity toGI:14423328 (or AAK62347) by Ralston et al. and Group 24 had 98.8%identity to GI:14423318 (or AAK62342) by Ralston et al. TABLE IV AminoAcid Sequence Identity Groups of Regions between Conserved Domains ofNicotiana p450 Genes 1 D208-AD9 (SEQ. ID. No. 224); D120-AH4 (SEQ. ID.No. 180); D121-AA8 (SEQ. ID. No. 182), D122-AF10 (SEQ. ID. No. 184);D103-AH3 (SEQ. ID. No. 222); D208-AC8 (SEQ. ID. No. 218); D-235-ABI(SEQ. ID. No. 246) 2 D244-AD4 (SEQ. ID. No. 250); D244-AB6 (SEQ. ID. No.274); D285-AA8; D285-AB9; D268-AE2 (SEQ. ID. No. 270) 3 D100A-AC3 (SEQ.ID. No. 168); D100A-BE2 4 D205-BE9 (SEQ. ID. No. 276); D205-BG9 (SEQ.ID. No. 202); D205-AH4 (SEQ. ID. No. 294) 5 D259-AB9 (SEQ. ID. No. 260);D257-AE4 (SEQ. ID. No. 268); D147-AD3 (SEQ. ID. No. 194) 6 D249-AE8(SEQ. ID. No. 256); D-248-AA6 (SEQ. ID. No. 254) 7 D233-AG7 (SEQ. ID.No. 266; D224-BD11 (SEQ. ID. No. 240); DAF10 8 D105-AD6 (SEQ. ID. No.172); D215-AB5 (SEQ. ID. No. 220); D135-AE1 (SEQ. ID. No. 190) 9D87A-AF3 (SEQ. ID. No. 216), D210-BD4 (SEQ. ID. No. 262) 10 D89-AB1(SEQ. ID. No. 150); D89-AD2 (SEQ. ID. No. 152); 163-AG11 (SEQ. ID. No.198); 163-AF12 (SEQ. ID. No. 196) 11 D267-AF10 (SEQ. ID. No. 296);D96-AC2 (SEQ. ID. No. 160); D96-AB6 (SEQ. ID. No. 158); D207-AA5 (SEQ.ID. No. 204); D207-AB4 (SEQ. ID. No. 206); D207-AC4 (SEQ. ID. No. 208)12 D98-AG1 (SEQ. ID. No. 164); D98-AA1 (SEQ. ID. No. 162) 13 D209-AA12(SEQ. ID. No. 212); D209-AA11; D209-AH10 (SEQ. ID. No. 214); D209-AH12(SEQ. ID. No. 232); D90a-BB3 (SEQ. ID. No. 154) 14 D129-AD10 (SEQ. ID.No. 188); D104A-AE8 (SEQ. ID. No. 170) 15 D228-AH8 (SEQ. ID. No. 244);D228-AD7 (SEQ. ID. No. 241), D250-AC11 (SEQ. ID. No. 258); D247-AH1(SEQ. ID. No. 252) 16 D128-AB7 (SEQ. ID. No. 186); D243-AA2 (SEQ. ID.No. 248); D125-AF11 (SEQ. ID. No. 228) 17 D284-AH5 (SEQ. ID. No. 298);D110-AF12 (SEQ. ID. No. 176) 18 D221-BB8 (SEQ. ID. No. 234) 19 D222-BH4(SEQ. ID. No. 236) 20 D134-AE11 (SEQ. ID. No. 230) 21 D109-AH8 (SEQ. ID.No. 174) 22 D136-AF4 (SEQ. ID. No. 278) 23 D237-AD1 (SEQ. ID. No. 226)24 D112-AA5 (SEQ. ID. No. 178) 25 D283-AC1 (SEQ. ID. No. 272) 26D110-AF12 (SEQ. ID. No. 176)

Example 12 Nicotiana Cytochrome P450 Clones Lacking One or More of theTobacco Cytochrome P450 Specific Domains

Four clones had high nucleic acid homology, ranging 90% to 99% nucleicacid homology, to other tobacco cytochrome genes reported in Table III.The four clones included D136-AD5, D138-AD12, D243-AB3 and D250-AC11.However, due to a nucleotide frameshift these genes did not contain oneor more of three C-terminus cytochrome p450 domains and were excludedfrom identity groups presented in Table III or Table IV.

The amino acid identity of one clone, D95-AG1, did not contain the thirddomain, GXRXC, used to group p450 tobacco genes in Table III or TableIV. The nucleic acid homology of this clone had low homology to othertobacco cytochrome genes. This clone represents a novel and differentgroup of cytochrome p450 genes in Nicotiana.

Example 13 Use of Nicotiana Cytochrome P450 Fragments and Clones inAltered Regulartion of Tobacco Properties

The use of tobacco p450 nucleic acid fragments or whole genes are usefulin identifying and selecting those plants that have altered tobaccophenotypes or tobacco constituents and, more importantly, alteredmetabolites. Transgenic tobacco plants are generated by a variety oftransformation systems that incorporate nucleic acid fragments or fulllength genes, selected from those reported herein, in orientations foreither down-regulation, for example anti-sense orientation, orover-expression for example, sense orienation. For over-expression tofull length genes, any nucleic acid sequence that encodes the entire ora functional part or amino acide sequence of the full-length genesdescribed in this invention are desired that are effective forincreasing the expression of a certain enzyme and thus resulting inphenotypic effect within Nicotiana. Nicotiana lines that are homozygouslines are obtained through a series of backcrossing and assessed forphenotypic changes including, but not limited to, analysis of endogenousp450 RNA, transcripts, p450 expressed peptides and concentrations ofplant metabolites using techniques commonly avaiable to one havingordinary skill in the art. The changes exhibited in the tobacco plansprovide information on the functional role of the selected gene ofinterest or are of a utility as a preffered Nicotiana plant species.

Example 14 Identification of Genes Induced in Ethylene Treated ConverterLines

High density oligonucleotide array technology, Affymetrix GeneChip®(Affymetrix Inc., Santa Clara, Calif.) array, was used for quantitativeand highly parallel measurements of gene expression. In using thistechnology, nucleic acid arrays were fabricated by direct synthesis ofoligonucleotides on a solid surface. This solid-phase chemistry is ableto produce arrays containing hundreds of thousands of oligonucleotideprobes packed at extremely high densities on a chip referred to asGeneChip®. Thousands of genes can be simultaneously screened from asingle hybridization. Each gene is typically represented by a set of11-25 pairs of probes depending upon size. The probes are designed tomaximize sensitivity, specificity, and reproducibility, allowingconsistent discrimination between specific and background signals, andbetween closely related target sequences.

Affymetrix GeneChip hybridization experiments involve the followingsteps: design and production of arrays, preparation of fluorescentlylabeled target from RNA isolated from the biological specimens,hybridization of the labeled target to the GeneChip, screening thearray, and analysis of the scanned image and generation of geneexpression profiles.

A. Designing and Custom Making Affymetrix GeneChip

A GeneChip CustomExpress Advantage Array was custom made by AffymetrixInc. (Santa Clara, Calif.). Chip size was 18 micron and array format was100-2187 that can accommodate 528 probe sets (11, 628 probes). Exceptfor GenBank derived nucleic acid sequences, all sequences were selectedfrom our previously identified tobacco clones and all probes were customdesigned. A total of 400 tobacco genes or fragments were selected to beincluded on the GeneChip. The sequences of oligonucleotides selectedwere based on unique regions of the 3′ end of the gene. The selectednucleic acid sequences consisted of 56 full length p450 genes and 71p450 fragments that were cloned from tobacco, described in (patentapplications). Other tobacco sequences included 270 tobacco ESTs whichwere generated from suppression subtraction library using Clontech SSHkit (BD Biosciences, Palo Alto, Calif.). Among these genes, someoligonucleotide sequences were selected from cytochrome P450 geneslisted in GenBank. Up to 25 probes were used for each full length geneand 11 probes for each fragment. A reduced number of probes were usedfor some clones due to the lack of unique, high quality probes.Appropriate control sequences were also included on the GeneChip®.

The probe Arrays were 25-mer oligonucleotides that were directlysynthesized onto a glass wafer by a combination of semiconductor-basedphotolithography and solid phase chemical synthesis technologies. Eacharray contained up to 100,000 different oligonucleotide probes. Sinceoligonucleotide probes are synthesized in known locations on the array,the hybridization patterns and signal intensities can be interpreted interms of gene identity and relative expression levels by the AffymetrixMicroarray Suite® software. Each probe pair consists of a perfect matcholigonucleotide and a mismatch oligonucleotide. The perfect match probehas a sequence exactly complimentary to the particular gene and thusmeasures the expression of the gene. The mismatch probe differs from theperfect match probe by a single base substitution at the center baseposition, which disturbs the binding of the target gene transcript. Themismatch produces a nonspecific hybridization signal or backgroundsignal that was compared to the signal measured for the perfect matcholigonucleotide.

B. Sample Preparation

Hybridization experiments were conducted by Genome Explorations, Inc.(Memphis, Tenn.). The RNA samples used in hybridization consisted of sixpairs of nonconverter/converter isogenic lines that were induced byethylene treatments. Samples included one pair of 4407-25/4407-33non-treated burly tobacco samples, three pairs of ethylene treated4407-25/4407-33 samples, one pair of ethylene treated dark tobacco NLMadole/181 and one pair of ethylene treated burly variety PBLB01/178.Ethylene treatment was as described in Example 1.

Total RNA was extracted from above mentioned ethylene treated andnon-treated leaves using a modified acid phenol and chloroformextraction protocol. Protocol was modified to use one gram of tissuethat was ground and subsequently vortexed in 5 ml of extraction buffer(100 mM Tris-HCl, pH 8.5; 200 mM NaCl; 10 mM EDTA; 0.5% SDS) to which 5ml phenol (pH5.5) and 5 ml chloroform was added. The extracted samplewas centrifuged and the supernatant was saved. This extraction step wasrepeated 2-3 more times until the supernatant appeared clear.Approximately 5 ml of chloroform was added to remove trace amounts ofphenol. RNA was precipitated from the combined supernatant fractions byadding a 3-fold volume of ETOH and 1/10 volume of 3M NaOAc (pH5.2) andstoring at −20° C. for 1 hour. After transferring to a Corex glasscontainer the RNA fraction was centrifuged at 9,000 RPM for 45 minutesat 4° C. The pellet was washed with 70% ethanol and spun for 5 minutesat 9,000 RPM at 4° C. After drying the pellet, the pelleted RNA wasdissolved in 0.5 ml RNase free water. The pelleted RNA was dissolved in0.5 ml RNase free water. The quality and quantity of total RNA wasanalyzed by denatured formaldehyde gel and spectrophotometer,respectively. The total RNA samples with 3-5 μg/ul were sent to Genomeexplorations, inc. to do the hybridization.

C. Hybridization, Detection and Data Output

The preparation of labeled cRNA material was performed as follows. Firstand second strand cDNA were synthesized from 5-15 μg of total RNA usingthe SuperScript Double-Stranded cDNA Synthesis Kit (Gibco LifeTechnologies) and oligo-dT24-T7 (5′-GGC CAG TGA ATT GTA ATA CGA CTC ACTATA GGG AGG CGG-3′) primer according to the manufacturer's instructions.

The cRNA was concurrently synthesized and labeled with biotinylated UTPand CTP by in vitro transcription using the T7 promoter coupled doublestranded cDNA as template and the T7 RNA Transcript Labeling Kit (ENZODiagnostics Inc.). Briefly, double stranded cDNA synthesized from theprevious steps were washed twice with 70% ethanol and resuspended in 22μl Rnase-free H2O. The cDNA was incubated with 4 μl of 10× each ReactionBuffer, Biotin Labeled Ribonucleotides, DTT, Rnase Inhibitor Mix and 2μl 20×T7 RNA Polymerase for 5 hr at 37° C. The labeled cRNA wasseparated from unincorporated ribonucleotides by passing through aCHROMA SPIN-100 column (Clontech) and precipitated at −20° C. for 1 hrto overnight.

Oligonucleotide array hybridization and analysis were performed asfollows. The cRNA pellet was resuspended in 10 μl Rnase-free H2O and10.0 μg was fragmented by heat and ion-mediated hydrolysis at 95° C. for35 mins in 200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc. Thefragmented cRNA was hybridized for 16 hr at 45° C. to HG_U95Av2oligonucleotide arrays (Affymetrix) containing ˜12,500 full lengthannotated genes together with additional probe sets designed torepresent EST sequences. Arrays were washed at 25° C. with 6×SSPE (0.9MNaCl, 60 mM NaH2PO4, 6 mM EDTA+0.01% Tween 20) followed by a stringentwash at 50° C. with 100 mM MES, 0.1M [Na+], 0.01% Tween 20. The arrayswere stained with phycoerythrein conjugated streptavidin (MolecularProbes) and the fluorescence intensities were determined using a laserconfocal scanner (Hewlett-Packard). The scanned images were analyzedusing Microarray software (Affymetrix). Sample loading and variations instaining were standardized by scaling the average of the fluorescentintensities of all genes on an array to constant target intensity (250)for all arrays used. Data Analysis was conducted using Microarray Suite5.0 (Affymetrix) following user guidelines. The signal intensity foreach gene was calculated as the average intensity difference,represented by [Σ(PM−MM)/(number of probe pairs)], where PM and MMdenote perfect-match and mismatch probes.

D. Data Analysis and Results

Twelve sets of hybridizations were successful as evidenced by theExpression Report generated using detection instruments from GenomeExplorations. The main parameters on the report included Noise, Scalefactor, background, total probe sets, number and percentage of presentand absent probe sets, signal intensity of housekeeping controls. Thedata was subsequently analyzed and presented using software GCOS incombination of other Microsoft software. Signal comparison betweentreatment pairs was analyzed. Overall data for all respective probescorresponding to genes and fragments of each different treatmentincluding replications were compiled and compiled expression data suchas call of the changes and signal log 2 ratio changes were analyzed.

A typical application of GeneChip technology is finding genes that aredifferentially expressed in different tissues. In the presentapplication, genetic expression variations caused by ethylene treatmentwere determined for pairs of converter and nonconverter tobacco linesthat included a 4407-25/4407-33 burley variety, PBLB01/178 burleyvariety, and a NL Madole/181 dark variety. These analyses detected onlythose genes whose expression is significantly altered due to biologicalvariation. These analyses employed the Fold change (signal ratio) as amajor criterion to identify induced genes. Other parameters, such assignal intensity, present/absent call, were also taken intoconsideration.

After analyzing the data for expression differences in converter andnonconverter pairs of samples for approximately 400 genes, the resultsbased on the signal intensities showed that only two genes, D121-AA8,and D120-AH4 and one fragment, D35-BG11, that is partial fragment ofD121-AA8, had reproducible induction in ethylene treated converter linesversus non-converter lines. To illustrate the differential expression ofthese genes, the data was represented as follows. As shown in Table V,the signal of a gene in a converter line, for example, burley tobaccovariety, 4407-33, was determined as ratio to the signal of a relatednonconverter isogenic line, 4407-25. Without ethylene treatment, theratio of converter to nonconverter signals for all genes approached1.00. Upon ethylene treatment, two genes, D121-AA8 and D120-AH4, wereinduced in converter lines relative to non-converter line as determinedby three independent analyses using isogenic burley lines. These geneshave very high homology to each other, approximately 99.8% or greaternucleic acid sequence homology. As depicted in Table V, their relativehybridization signals in converter varieties ranged from approximately 2to 12 fold higher in converter lines than the signals in theirnon-converter counterparts. In comparison, two actin-like controlclones, internal controls, were found not to be induced in converterlines based on their normalized ratios. In addition, a fragment(D35-BG11), whose sequence in coding region is entirely contained inboth D121-AA8 and D120-AH4 genes, was highly induced in the same samplesof paired isogenic converter and nonconverter lines. Another isogenicpair of burley tobacco varieties, PBLB01 and 178, was shown to have thesame genes, D121-AA8 and D120-AH4, induced in converter samples underethylene induction. Furthermore, D121-AA8 and D120-AH4 genes werepreferentially induced in converter lines of isogenic dark tobaccopairs, NL Madole and 181, demonstrating that ethylene induction of thesegenes in converter lines was not limited to burley tobacco varieties. Inall cases, the D35-BG11 fragment was the most highly induced inconverter relative to nonconverter paired lines. TABLE V A Comparison ofClone Induction in Ethylene Treated Converter and Non-Converter Lines NoEthylene Treated Ethylene Treated Ethylene Treated Ethylene TreatedEthylene Treated Treatment Burley Exp 1 Burley Exp 2 Burley Exp 3 BurleyExp 4 Dark 33:25 33:25 Et:No* 33:25 Et:No 33:25 Et:No 33:25 Et:No 181:NLEt:No Clones Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio Ratio RatioRatio Induced D121-AA8 1.03 2.20 2.14 13.25 12.90 5.31 5.15 12.56 12.1917.06 16.60 D120-AH4 1.44 2.74 1.90 18.33 12.74 4.13 2.87 10.87 7.5511.76 8.17 Control Actin-Like I 1.18 1.17 0.99 0.88 0.74 0.86 0.73 0.670.57 1.20 1.02 (5′) Actin-Like I 1.09 1.23 1.12 0.89 0.81 1.18 0.11 0.860.79 1.02 0.93 (3′)*normalized Ratio.

Example 15 Ethylene Induction of Microsomal Nicotine Demethylase inTobacco Converter Lines

Biochemical analyses of demethylase enzymatic activity in microsomalenriched fractions of ethylene treated and non-treated pairs ofconverter and non-converter tobacco lines were performed as follows.

A. Preparation of Microsomes

Microsomes were isolated at 4° C. Tobacco leaves were extracted in abuffer consisting of 50 mM N-(2-hydrooxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 7.5, 3 mMDL-Dithiothreitol (DTT) and Protease Inhibitor Cocktail (Roche) at 1tablet/50 ml. The crude extract was filtered through four layers ofcheesecloth to remove undisrupted tissue, and the filtrate wascentrifuged for 20 min at 20,000×g to remove cellular debris. Thesupernatant was subjected to ultracentrifugation at 100,000×g for 60 minand the resultant pellet contained the microsomal fraction. Themicrosomal fraction was suspended in the extraction buffer and appliedto an ultracentrifugation step where a discontinuous sucrose gradient of0.5 M sucrose in the extraction buffer was used. The purified microsomeswere resuspended in the extraction buffer supplemented with 10% (w/v)glycerol as cryoprotectant. Microsomal preparations were stored in aliquid nitrogen freezer until use.

B. Protein Concentration Determination

Microsomal proteins were precipitated with 10% Trichloroacetic Acid(TCA) (w/v) in acetone, and the protein concentrations of microsomeswere determined using RC DC Protein Assay Kit (BIO-RAD) following themanufacturer's protocol._(—)

3) Nicotine Demethylase Activity Assay

DL-Nicotine (Pyrrolidine-2-¹⁴C) was obtained from Moravek Biochemicalsand had a specific activity of 54 mCi/mmol. Chlorpromazine (CPZ) andoxidized cytochrome c (cyt. C), both P450 inhibitors, were purchasedfrom Sigma. Reduced form of nicotinamide adenine dinucleotide phosphate(NADPH) is the typical electron donor for cytochrome P450 via theNADPH:cytochrome P450 reductase. NADPH was omitted for controlincubation. Routine enzyme assay consisted of microsomal proteins(around 2 mg/ml), 6 mM NADPH, 55 μM ¹⁴C labeled nicotine. Theconcentration of CPZ and Cyt. C, when used, was 1 mM and 100 μM,respectively. The reaction was carried at 25° C. for 1 hour and wasstopped with addition of 300 μl methanol to each 25 μl reaction mixture.After spinning, 20 μl of the methanol extract was separated with areverse-phase High Performance Liquid Chromatography (HPLC) system(Agilent) using an Inertsil ODS-3 3μ (150×4.6 mm) column from Varian.The isocratic mobile phase was the mixture of methanol and 50 mMpotassium phosphate buffer, pH 6.25, with ratio of 60:40 (v/v) and theflow rate was 1 ml/min. The nornicotine peak, as determined bycomparison with authentic non-labeled nornicotine, was collected andsubjected to 2900 tri-carb Liquid Scintillation Counter (LSC) (PerkinElmer) for quantification. The activity of nicotine demethylase iscalculated based on the production of ¹⁴C labeled nornicotine over 1hour incubation.

Samples were obtained from pairs of Burley converter (line 4407-33) andnon-converter (line 4407-25) tobacco lines that were ethylene treated ornot. All untreated samples did not have any detectable microsomalnicotine demethylase activity. In contrast, microsomal samples obtainedfrom ethylene treated converter lines were found to contain significantlevels of nicotine demethylase activity. The nicotine demethylaseactivity was shown to be inhibited by P450 specific inhibitorsdemonstrating the demethylase activity was consistent to a P450microsomal derived enzyme. A typical set of enzyme assay resultsobtained for the burley converter tobacco line is shown in the Table VI.In contrast, sample derived from ethylene treated nonconverter tobaccodid not contain any nicotine demethylase activity. These resultsdemonstrated that nicotine demethylase activity was induced upontreatment with ethylene in converter lines but not in the correspondingisogenic nonconverter line. Similar results were obtained for anisogenic dark tobacco variety pair, where microsomal nicotinedemethylase activity was induced in converter lines and not detectablein nonconverter paired lines. Together these experiments demonstratedthat microsomal nicotine demethylase activity is induced upon ethylenetreatment in converter lines while not in paired isogenic nonconverterlines. Those genes that are P450 derived genes and are preferentiallyinduced in converter lines relative to paired non-converter lines arecandidate genes to encode the nicotine demethylase enzyme. TABLE VIDEMETHYLASE ACTIVITY IN MICROSOMES OF ETHYLENE INDUCED BURLEY CONVERTERAND NON CONVERTER LINES Microsomes + Microsomes + with Micro- 1 mM 100μM Microsomes − Sample somes chlorpromazine cytochrome C NADPH Converter8.3 ± 0.4 0.01 ± 0.01 0.2 ± 0.2 0.4 ± 0.4 pkat/mg pkat/mg pkat/mgpkat/mg protein protein protein protein Non- Not Not Detected NotDetected Not Detected Converter Detected

Example 16 Functional Identification of D121-AA8 as Nicotine Demethylase

The function of the candidate clone (D121-AA8), was confirmed as thecoding gene for nicotine demethylase, by assaying enzyme activity ofheterologously expressed P450 in yeast cells.

1. Construction of Yeast Expression Vector

The putative protein-coding sequence of the P450-encoding cDNA (121AA8),was cloned into the yeast expression vector pYeDP60. Appropriate BamHIand MfeI sites (underlined) were introduced via PCR primers containingthese sequences either upstream of the translation start coden (ATG) ordownstream of the stop coden (TAA). The MfeI on the amplified PCRproduct is compatible with the EcoRI site on the vector. The primersused to amplify the 121AA8 cDNA were5′-TAGCTACGCGGATCCATGCTTTCTCCCATAGAAGCC-3′ and5′-CTGGATCACAATTGTTAGTGATGGTGATGGTGATGCGATCCTCTATAAAGCTCAGGTGCCAGGC-3′.A segment of sequence coding nine extra amino acids at the C-terminus ofthe protein, including six histidines, was incorporated into the reverseprimer. This facilitates the expression of 6×His tagged P450 uponinduction. PCR products were ligated into pYeDP60 vector after enzymedigestions in the sense orientation with reference to the GAL10-CYC1promoter. Constructs were verified by enzyme restrictions and DNAsequencing.

2. Yeast Transformation

The WAT11 yeast line, modified to express Arabidopsis NADPH-cytochromeP450 reductase ATR1, was transformed with the construct pYeDP60-P450cDNA plasmids. Fifty micro-liter of WAT11 yeast cell suspension wasmixed with ˜1 μg plasmid DNA in a cuvette with 0.2-cm electrode gap. Onepulse at 2.0 kV was applied by an Eppendorf electroporator (Model 2510).Cells were spread onto SGI plates (5 g/L bactocasamino acids, 6.7 g/Lyeast nitrogen base without amino acids, 20 g/L glucose, 40 mg/LDL-tryptophan, 20 g/L agar). Transformants were confirmed by PCRanalysis performed directly on randomly selected colonies.

3. P450 Expression in Transformed Yeast Cells

Single yeast colonies were used to inoculate 30 mL SGI media (5 g/Lbactocasamino acids, 6.7 g/L yeast nitrogen base without amino acids, 20g/L glucose, 40 mg/L DL-tryptophan) and grown at 30° C. for about 24hours. An aliquot of this culture was diluted 1:50 into 1000 mL of YPGEmedia (10 g/L yeast extract, 20 g/L bacto peptone, 5 g/L glucose, 30ml/L ethanol) and grown until glucose was completely consumed asindicated by the colorimetric change of a Diastix urinalysis reagentstrip (Bayer, Elkhart, Ind.). Induction of cloned P450 was initiated byadding DL-galactose to a final concentration of 2%. The cultures weregrown for an additional 20 hours before used for in vivo activity assayor for microsome preparation.

WAT11 yeast cells expressing pYeDP60-CYP71D20 (a P450 catalyzing thehydroxylation of 5-epi-aristolochene and 1-deoxycapsidiol in Nicotianatabacum) were used as control for the P450 expression and enzymeactivity assays.

4. In Vivo Enzyme Assay

The nicotine demethylase activity in the transformed yeast cells wereassayed by feeding of yeast culture with DL-Nicotine(Pyrrolidine-2-¹⁴C). To 75 μl of the galactose induced culture ¹⁴Clabeled nicotine (54 mCi/mmol) was added to a final concentration of 55μM. The assay culture was incubated with shaking in 14 ml polypropylenetubes for 6 hours and was extracted with 900 μl methanol. Afterspinning, 20 μl of the methanol extract was separated with an rp-HPLCand the nornicotine fraction was quantitated by LSC.

The control culture of WAT11 (pYeDP60-CYP71D20) did not convert nicotineto nornicotine, showing that the WAT11 yeast strain does not containendogenous enzyme activities that can catalyze the step of nicotinebioconversion to nornicotine. In contrast, yeast expressing 121AA8 geneproduced detectable amount of nornicotine, indicating the nicotinedemethylase activity of this P450 enzyme.

5. Yeast Microsome Preparation

After induction by galactose for 20 hours, yeast cells were collected bycentrifugation and washed twice with TES-M buffer (50 mM Tris-HCl, pH7.5, 1 mM EDTA, 0.6 M sorbitol, 10 mM 2-mercaptoethanol). The pellet wasresuspended in extraction buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.6M sorbitol, 2 mM 2-mercaptoethanol, 1% bovine serum album, ProteaseInhibitor Cocktail (Roche) at 1 tablet/50 ml). Cells were then brokenwith glass beads (0.5 mm in diameter, Sigma). Cell extract wascentrifuged for 20 min at 20,000×g to remove cellular debris. Thesupernatant was subjected to ultracentrifugation at 100,000×g for 60 minand the resultant pellet contained the microsomal fraction. Themicrosomal fraction was suspended in TEG-M buffer (50 mM Tris-HCl, pH7.5, 1 mM EDTA, 20% glycerol and 1.5 mM 2-mercaptoethanol) at proteinconcentration of 1 mg/mL. Microsomal preparations were stored in aliquid nitrogen freezer until use.

6. Enzyme Activity Assay in Yeast Microsomal Preparations

Nicotine demethylase activity assays with yeast microsomal preparationswere performed in the same way as with microsomal preparations fromtobacco leaves (EXAMPLE 15) except that the protein concentrations wereconstant at 1 mg/mL.

Microsomal preparations from control yeast cells expressing CYP71D20 didnot have any detectable microsomal nicotine demethylase activity. Incontrast, microsomal samples obtained from yeast cells expressing 121AA8gene showed significant levels of nicotine demethylase activity. Thenicotine demethylase activity had requirement for NADPH and was shown tobe inhibited by P450 specific inhibitors, consistent to the P450 beinginvestigated. A typical set of enzyme assay results obtained for theyeast cells is shown in the Table VII. TABLE VII DEMETHYLASE ACTIVITY INMICROSOMES OF YEAST CELLS EXPRESSING 121AA8 AND CONTROL P450Microsomes + with Microsomes + 1 mM 100 μM Sample Microsomeschlorpromazine cytochrome C Microsomes − NADPH D121-AA8 10.8 ± 1.2* 1.4± 1.3 2.4 ± 0.7 0.4 ± 0.1 pkat/mg pkat/mg pkat/mg pkat/mg proteinprotein protein protein Control Not Not Not Not Detected (CYP71D20)Detected Detected Detected*Average results of 3 replicates.

Together these experiments demonstrated that the cloned full length geneD121-AA8 encodes cytochrome P450 protein that catalyzes the conversionof nicotine to nornicotine when expressed in yeast.

1. An isolated nucleic acid molecule from Nicotiana, wherein saidnucleic acid molecule is SEQ. ID. No.:
 181. 2. An isolated nucleic acidmolecule from Nicotiana, wherein said nucleic acid molecule has at least81% sequence identity to SEQ. ID. No.:
 181. 3. An isolated nucleic acidmolecule from Nicotiana, wherein said nucleic acid molecule has at least91% sequence identity to SEQ. ID. No.:
 181. 4. An isolated protein fromNicotiana, wherein said protein comprises SEQ. ID. No.:
 182. 5. Anisolated protein from Nicotiana, wherein said protein has at least 80percent sequence identity to SEQ. ID. No.:
 182. 6. An isolated proteinfrom Nicotiana, wherein said protein has at least 90 percent sequenceidentity to SEQ. ID. No.:
 182. 7. A transgenic plant, wherein saidtransgenic plant comprises the nucleic acid molecule of claim 1, 2 or 3.8. The transgenic plant of claim 7, wherein said plant is a tobaccoplant.
 9. A method of producing a transgenic plant, wherein said methodcomprises the steps of: (i) operably linking said nucleic acid moleculeof claim 1, 2 or 3 with a promoter functional in said plant to create aplant transformational vector; (ii) transforming said plant with saidplant transformational vector of step (i); (iii) selecting a plant celltransformed with said transformation vector; and (iv) regenerating atransformation plant from said transformed plant cell.
 10. The method ofclaim 9, wherein said nucleic acid molecule is in an antisenseorientation.
 11. The method of claim 9, wherein said nucleic acidmolecule is in a sense orientation.
 12. The method of claim 9, whereinsaid nucleic acid molecule is in a RNA interference orientation.
 13. Themethod of claim 9, wherein said nucleic acid molecule is expressed as adouble stranded RNA molecule.
 14. The method of claim 9, wherein saidtransgenic plant is a tobacco plant.
 15. A method of selecting a plantcontaining a nucleic acid molecule, wherein said plant is analyzed forthe presence of a nucleic acid sequence of claim 1, 2 or
 3. 16. Themethod of selecting a plant of claim 15, wherein said plant is analyzedby DNA hybridization.
 17. The method of selecting a plant of claim 16,wherein said DNA hybridization is Southern blot analysis.
 18. The methodof selecting a plant of claim 16, wherein said DNA hybridization isNorthern blot analysis.
 19. The method of selecting a plant of claim 15,wherein said plant is analyzed by PCR detection.
 20. The method of claim15, wherein said plant is a tobacco plant.
 21. A method of increasing ordecreasing nornicotine levels in a plant, wherein said method comprisesthe steps of: (i) operably linking said nucleic acid molecule of claim1, 2 or 3 with a promoter functional in said plant to create a planttransformational vector; (ii) transforming said plant with said planttransformational vector of step (i); (iii) selecting a plant celltransformed with said transformation vector; and (iv) regenerating atransformation plant from said transformed plant cell.
 22. The method ofclaim 21, wherein said nucleic acid molecule is in an antisenseorientation.
 23. The method of claim 21, wherein said nucleic acidmolecule is in a sense orientation.
 24. The method of claim 21, whereinsaid nucleic acid molecule is in a RNA interference orientation.
 25. Themethod of claim 21, wherein said nucleic acid molecule is expressed as adouble stranded RNA molecule.
 26. The method of claim 21, wherein saidtransgenic plant is a tobacco plant.